Systems and methods to estimate nutritional needs of human and other patients

ABSTRACT

Systems, techniques and methods for estimating the metabolic state or flux, e.g., the body energy state (“BES”) of a patient, are disclosed. The BES provides deep insight into the nutritional needs of the patient, thus allowing for a sort of exquisite glycemic control with regard to the patient. The invention discloses systems and methods for estimating fractional gluconeogenesis, which is the % of glucose production that comes from gluconeogenesis (“GNG”), as opposed to glycogenolysis (“GLY”), the other form of glucose production. Nutritional formulations, materials, cocktails and methods for feeding patients by parenteral and other means are disclosed. The amount, type and rate of such nutritional feeding are typically based upon the above estimating. The invention discloses formulations that contain labels, such as deuterium, for medical diagnostics, such as for estimating BES and fractional gluconeogenesis. The invention is suitable for any sort of patient, including those who are injured, such as with traumatic brain injury, ill, or have other conditions that stress the metabolic system.

APPLICATION PRIORITY DATA

The current patent application claims priority to U.S. provisionalpatent application 61/795,819 filed on Oct. 25, 2012 by Horning andBrooks and is a continuation of U.S. patent application Ser. No.13/903,929 filed on May 28, 2013 by Horning and Brooks titled “SYSTEMSAND METHODS TO ESTIMATE NUTRITIONAL NEEDS OF HUMAN AND OTHER PATIENTS”,U.S. patent application Ser. No. 13/903,936 filed on May 28, 2013 byHorning and Brooks titled “FORMULATIONS AND METHODS TO PROVIDE NUTRITIONTO HUMAN AND OTHER PATIENTS”, and U.S. patent application Ser. No.13/903,939 filed on May 28, 2013 by Horning and Brooks titled“FORMULATIONS CONTAINING LABELS FOR MEDICAL DIAGNOSTICS”, and is relatedto U.S. patent application ______ filed on Aug. 2, 2013 by Horning andBrooks titled “FORMULATIONS AND METHODS TO PROVIDE NUTRITION TO HUMANAND OTHER PATIENTS”, and U.S. patent application ______ filed on Aug. 2,2013 by Horning and Brooks titled “FORMULATIONS CONTAINING LABELS FORMEDICAL DIAGNOSTICS”, which are incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present disclosure generally relates to the field of medicaltreatment. More specifically, the invention presents systems and methodsto ascertain the metabolic state and nutritional needs of a patient,which can be thought of as the body energy state (“BES”) of the patient.Assessment of the BES of the patient is critical information to treatand nourish (feed) the patient appropriately. Such assessment is basedon ongoing and dynamic estimates of the biomarker fractionalgluconeogenesis, which is the % of body glucose production that comesfrom gluconeogenesis. Methods, systems and materials for patientnutritional treatment and feeding based on estimation of this biomarkerare also provided.

BACKGROUND OF THE INVENTION

Glucose is a basic fuel of the human body (as well as of many otherorganisms) and is delivered throughout the body through the blood. Therate of glucose production, also referred to as glucose rate ofappearance and glucose Ra, is about 2-3 mg/min/kg of body weight in ahealthy person while at rest, and can be as high as 8 mg/min/kg or moreunder stress such as exercise or illness. Pyruvate and lactate, whichare both gluconeogenic precursors and products of glucose catabolism,are also basic fuels of the human body and other organisms.

Glucose, a six-carbon (hexose) sugar, is an essential fuel energy sourcefor several vitally important organs and tissues in the body, includingthe brain and nerves that require a continuous glucose supply, includingafter injury. Not surprisingly, glucose is an important and tightlyregulated metabolite.

Glucose Ra should not be confused with blood concentration of glucose,also called [glucose]. The latter is a simple measure of the totalamount of glucose in the blood, as opposed to the rate of production.The [glucose] is a common measurement taken from a blood samples, as instandard doctor office visits and home diabetes diagnostics. This valuecan vary significantly in resting individuals, but generally averagesabout 90-100 mg/dl blood or 5.5 mM. Physiologically, glucose can appearin the blood of a person by three major means: delivery from ingestedcarbohydrate-containing foods, hepatic glycogenolysis (“GLY”), andgluconeogenesis (“GNG”) (hepatic and renal). The recommended dietaryallowance for carbohydrate-containing foods is about 130 g/day, a valuedetermined to be the minimal daily brain glucose requirement (8) (notethat non-patent literature citations are made as numbers in parentheses,and the corresponding references are listed at the end of thisspecification). Hence, dietary carbohydrate and total nutrientinadequacy will reflexively cause increased GLY and GNG to maintainglucose requirements for the brain, other tissues with high glucoseneeds (nerves, red blood cells, kidneys) and the body in general.

Glucose production occurs by GLY and GNG. It is generally better if themajority of glucose production is from GLY. This is because GLY is anefficient process of glucose production, in that it is simple breakdownof glycogen, a glucose polymer stored mainly in the muscles, liver andkidneys. Normally, at rest, in a nourished state, most glucose isproduced by GLY (typically over 75%). This number can decrease understress such as exercise or illness, as the body needs to produce moreglucose than can be provided by GLY.

Gluconeogenesis (“GNG”) describes essentially all pathways for producingglucose other than glycogenolysis (“GLY”). GNG produces glucose fromcarbon substrates such as pyruvate, lactate, glycerol, and gluconeogenicamino acids, among others. These can be termed GNG precursors. GNG isless efficient than GLY in terms of glucose produced per unit of storedenergy because of the more complex pathways needed to produce it. Sinceit is less efficient than GLY, it is generally not preferred by thebody, but can be used to produce glucose as needed. GNG is lessefficient than GLY in other ways as well. The work of raising a GNGprecursor to the level of glucose 6-phosphate and glucose requiressignificant energy input, and important body constituents such as leanbody mass, muscle are often degraded to provide precursor materials forthe process. GNG also may be used to access glycogen stored elsewhere inthe body instead of direct conversion of that glycogen to glucose.

The current art in the measurement of metabolic state and treatment hasat least two significant categories of problems. One is that nobiomarker measurements, either alone or in combination, are used in thecurrent art to give an accurate picture of the overall BES of a patient.To the degree that measurements are made in the current art, such aswith [glucose], they are inadequate indicators of the BES.

The biomarker [glucose], is well known in the art and simple to assessfrom a blood test. While a large shift (either low or high) in [glucose]can be cause for concern and inform the type of feeding the patientreceives, it does not provide a good indicator of the BES of a patient,especially within its typical ranges. Indeed, the maintenance of bloodglucose homeostasis is a top physiological priority, and there arediverse and redundant body mechanisms to maintain blood [glucose]. Thusa normal [glucose] may belie metabolic stresses that are going on, withthe body working very hard to maintain [glucose]. Among those mechanismsare GNG, a critically important process about which the blood [glucose]measurement provides no direct information.

Another biomarker, glucose rate of appearance (“Ra”), gives only aslightly better indicator of the BES of the patient. A high glucose Ra,for example, indicates that the patient may be experiencing a stress(such as injury, exercise or starvation) that has induced a high glucoseproduction. While this is a somewhat useful, there is need for abiomarker that is a more precise indicator of BES. In addition,determination of glucose Ra is complex, time consuming and costly. Itrequires labeled glucose to be given to the patient, typically glucosewith deuterium (typically noted as simply D or ²H as opposed to merelyH, hydrogen), or carbon 13 (¹³C), and comparison of labeled andnon-labeled glucose (the latter produced by the glucose pathways) todetermine Ra (80).

The complex, costly and time-consuming process of determining glucose Rawith stable isotopes of H (typically deuterium) or ¹³C-glucose is welldescribed in the literature (2, 26, 55). It is typically done asfollows. Control subjects or patients receive a primed continuousinfusion of [6,6-²H]glucose, i.e., D₂-glucose, glucose with twodeuteriums on carbon number 6 (C-6) diluted in 0.9% sterile saline andtested for pyrogenicity and sterility prior to infusion. To hastenachievement of a constant blood isotopic enrichment, a priming bolus ofperhaps about 125 times the continuous per minute infusion rate, orabout 250 mg D₂-glucose, is infused over several min prior tocommencement of a continuous tracer infusion of 2.0 mg·min⁻¹ D₂-glucose.In this manner, isotopic equilibration in the blood can be achieved in60-90 min (about half the time to isotopic equilibration in blood if apriming tracer dose is not given).

To verify when isotopic equilibration has been achieved, several ml ofblood is drawn serially. Verification can be done by mixing in severalvolumes of 6-8% perchloric acid (“PCA”), and the deproteinizedsupernatant analyzed by means of forming a penta-acetate derivativefollowed by analysis using gas chromatography/mass spectrometry(“GC/MS”).

For simultaneous concentration analysis, known amounts of a labeledinternal standard, such as uniformly labeled glucose, where each carbonof the glucose is labeled, by for example, the carbon 13 isotope, thusnoted [U-¹³C]glucose, is used. The glucose molecule thus has anincreased mass of about 6 atomic units (“au”) (m+6). This labeledglucose is added to the supernatant of control subject or patientsamples collected in perchloric acid. To separate glucose, samples areneutralized with 2N KOH and transferred to cation resin, ion exchangecolumns such as 50W-X8 (from Bio-Rad Laboratories). Glucose is elutedfirst with doubly deionized H₂O (the anions, and cations, by contrast,are retained on the column).

The glucose ion-exchange effluent is reduced by lyophilization andderivatized by resuspending the lyophilized sample in a small amount(e.g., 1 ml) of methanol, a small amount [e.g., 200 microliter (0)] istransferred to a 2 ml microreaction vial and dried under N₂ gas. A smallamount (e.g., 100 μl) of a 2:1 acetic anhydride-pyridine solution isadded to each sample vial and heated at 60° C. for 10 min. Samples areagain dried under N₂ gas, resuspended in a small amount (e.g., 200 μl)of ethyl acetate, and transferred to micro vials for analysis.

Glucose isotopic enrichment (“IE”) is determined by GC/MS, for instancewith a GC model 6890 series and MS model 5973N, from AgilentTechnologies) of the penta-acetate derivative, where methane is used forselected ion monitoring of mass-to-charge ratios (m/z) 331 (non-labeledglucose), 332 (M+1 isotopomer, [1-¹³C]glucose), 333 (M+2 isotopomer,D₂-glucose), and 337 (M+6 isotopomer, [U-¹³C]glucose, the internalstandard). Whole blood glucose concentration is determined by abundanceratios of 331/337. Selected ion abundances are compared against externalstandard curves for calculation of concentration and isotopicenrichment.

Therefore there is a need in the art for a biomarker that is a goodindicator, by itself, of BES, as well as simple and effective methods ofestimating that biomarker.

SUMMARY OF THE INVENTION

The invention presents systems and methods to ascertain the metabolicstate and nutritional needs of a patient. Such assessment is based onongoing and dynamic estimates of the biomarker fractionalgluconeogenesis, which is the % of body glucose production that comesfrom gluconeogenesis. Methods, systems and materials for patientnutritional treatment and feeding based on estimation of this biomarkerare also provided. The invention includes, but is not limited to thefollowing, with some variation.

According to an embodiment of the present disclosure, the inventionprovides a method for estimating the fractional gluconeogenesis of apatient, administering a label to the patient, taking a blood samplefrom the patient, analyzing glucose or a glucose derivative from theblood sample, obtaining a value for fractional gluconeogenesis based onabundance from one or more mass spectra, obtaining a value forfractional gluconeogenesis plus glycogenolysis from one or more massspectra, and estimating fractional gluconeogenesis.

According to an embodiment of the present disclosure, the inventionprovides a method for providing nutritional support to a patient,including administering a label to the patient, taking a blood samplefrom the patient, analyzing glucose or a glucose derivative from theblood sample, obtaining a value for fractional gluconeogenesis based onabundance from one or more mass spectra, obtaining a value forfractional gluconeogenesis plus glycogenolysis from one or more massspectra, using the value to create to estimate fractionalgluconeogenesis, and administering a parenteral nutritive formulation tothe patient based upon the fractional gluconeogenesis estimate. Thelabel may be deuterium.

According to an embodiment of the present disclosure, the inventionprovides a method for estimating the fractional gluconeogenesis of apatient, the method including, administering a label to the patient,estimating the fraction of body water that has been labeled, using thisestimate to create a baseline for the amount of total glucoseproduction, estimating an amount of glucose production only fromgluconeogenesis by measuring the label, and estimating the patient'sfractional gluconeogenesis.

The methods can include a water labeled with deuterium, wherein lessthan about 1% of the body water is labeled, wherein the body water islabeled with an initial bolus, wherein the body water is continuallylabeled by ongoing infusion of labeled water, wherein the glucosederivative is a penta-acetate glucose molecule with molecular weight ofabout 390, wherein part of the estimation is based on the abundance ofthe label on one or more of glucose carbons 1, 3, 4, 5, 6, wherein partof the estimation is based on the abundance of the label on glucosecarbon 2, wherein glucose Ra is estimated to further provide an estimateof absolute rate of GNG, and using a correction factor to correct forthe fraction of the molecule that exists in a state that includes thelabel. The method provides for molecule analysis in a gas chromatographmass spectrometer. The method provides, upon estimating the fractionalgluconeogenesis, the patient is administered a parenteral nutritiveformulation, wherein the formulation may contain MCC or GNG precursor orboth, pyruvate or lactate or both, wherein the formulation isadministered or increased if the estimated fractional GNG is above about25% or 35%, wherein the formulation is stopped or decreased if theestimated fractional GNG is below about 15% or 20%.

According to an embodiment of the present disclosure, the inventionprovides a parenteral nutritive formulation for feeding a patient todecrease or stabilize fractional gluconeogenesis, including water andMCC or GNG precursor or both. It also provides a parenteral nutritiveformulation for feeding a patient with injury or illness, includingwater and MCC or GNG precursor or both. It also provides parenteralnutritive formulation for feeding a patient to decrease or stabilizefractional gluconeogenesis, the formulation including water and lactateor pyruvate or both, and one or more salts, wherein the formulation hasan osmolality less than about 310 mOsm.

The formulations may also include one or more salts, one or more of Na⁺,K⁺, Ca⁺⁺, Mg⁺⁺, and H₂PO₄— a label such as deuterium, have an osmolalityof less than about 310 mOsm, where one of the MCCs or GNGs is lactate orpyruvate or both, where one of the MCCs or GNGs is an amino acid whereone of the MCCs or GNGs is a GNG precursor that naturally occurs in thebody, where one of the MCCs or GNGs is a compound that does notnaturally occur in the body but that can be used as a GNG precursor,where one of the MCCs or GNGs is glycerol or glycerol tri-lactate. Theformulation may be administered at a rate of about 3 mg/kg/min, where kgis kg of patient body weight and 3 mg is the amount of MCC or GNG in theformulation, may be administered at a rate of about 50 micro moles perkg of body weight per minute (μMoles/kg/min), where kg is kg of patientbody weight and 50 μM is the amount of MCC or GNG in the formulation,administered or increased if estimated fractional GNG is above about 25%or 35%, or decreased or stopped if estimate of fractionalgluconeogenesis is below about 20% or 15%.

The formulations may include a label such as deuterium and one or moresalts. They may contain or more of the following: Na+, K⁺, Ca⁺⁺, Mg⁺⁺,and H₂PO₄—. They may have Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, and H₂PO₄— provided inthe ratio of about 145, 4, 2.5, 1.5, and 1.0 respectively. They may haveMCC or GNG precursor or both. The formulation may have an osmolality ofless than about 310 mOsm. The formulation may have an MCCs or GNGs thatis lactate or pyruvate or both, an amino acid, a GNG precursor thatnaturally occurs in the body, a compound that does not naturally occurin the body but that can be used as a GNG precursor, glyceroltri-lactate or arginyl lactate. The formulation may be administered at arate of about 3 mg/kg/min, where kg is kg of patient body weight and 3mg is the amount of MCC or GNG precursor in the formulation and may beadministered or increased if estimated fractional GNG is above about 25%or 35%, or decreased or stopped if estimate of fractionalgluconeogenesis is below about 20% or 15%. The formulations may beparenteral, used to estimate fractional GNG, used to stabilize ordecrease fractional GNG. The label may be incorporated into glucose. Thelabel may be differentially incorporated into glucose depending onwhether it is incorporated via the gluconeogenesis pathway or via theglycogenolysis pathway.

The nutritive formulations may include deuterium, lactate or pyruvate orboth, and one or more salts, may have an osmolality of less than about310 mOsm, may have one more of the following: Na+, K⁺, Ca⁺⁺, Mg⁺⁺, andH₂PO₄—, may be parenteral. The nutritive formulation may be used todecrease or stabilize fractional gluconeogenesis, and include deuterium,lactate or pyruvate or both, and one or more salts, may have anosmolality of less than about 310 mOsm, and may have one more of thefollowing: Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, and H₂PO₄—.

In all of the systems and methods, various labels may be used, includingdeuterium, such as in deuterium oxide (water), and sometimes at aconcentration of less than about 1% of the water. The formulations maybe enteral or parenteral.

BRIEF DESCRIPTION OF THE DRAWINGS

The described techniques and mechanisms, together with other features,embodiments, and advantages of the present disclosure, may best beunderstood by reference to the following description taken inconjunction with the accompanying drawings, which illustrate variousembodiments of the present techniques and mechanisms. In the drawings,structural elements having the same or similar functions are denoted bylike reference numerals.

FIG. 1 is a standard chemical representation/illustration of themolecule glucose, approximate molecular weight (“MW”) 180, in thebiologically dominant alpha-D-glucose confirmation.

FIG. 2 is a standard chemical representation/illustration of themolecule glucose, with all seven hydrogens replaced with deuteriums (aslabels).

FIG. 3 shows the penta-acetate glucose derivative, approximate MW 390,that is part of the mass spectra analysis method, in a preferredembodiment of the invention.

FIG. 4 shows a fragment of the penta-acetate glucose derivative with allof the hydrogens of interest still on the molecule, approximate MW 331,as well as the same molecule with different isotopes (MW 332, etc.)

FIG. 5 shows a fragment of the penta-acetate glucose derivative,approximate MW 271, as well as the same molecule with different isotopes(MW 272, etc.)

FIG. 6 shows another fragment of the penta-acetate glucose derivative,approximate MW 169, as well as the same molecule with different isotopes(MW 170, etc.)

FIG. 7 shows a schematic mass spectrum focusing on the MW 331 andrelated ions.

FIG. 8 shows a schematic mass spectrum focusing on the MW 169 andrelated ions.

FIG. 9 shows a schematic mass spectrum showing the MW 169, 271, 331 andrelated ions.

FIG. 10 shows an actual GC/MS spectrum of the current invention, showingpeaks corresponding to the selected ion monitoring (“SIM”) of the MW 331and 332 ions, where the intensity of the signal is calculated byintegrating the areas under the peaks.

DETAILED DESCRIPTION OF THE INVENTION

Gluconeogenesis (“GNG”)

That superior biomarker for assessing the BES of a patient, as disclosedin the present invention, is fractional GNG, that is, the % of totalglucose production that comes from GNG.

Various embodiments of the present disclosure provide systems andmechanisms for estimating fractional GNG in a patient. The disclosedinvention includes systems and methods for determining the nutritionstate and needs of the patient also based on the absolute rate of GNGand the rate of glucose appearance, among other measures. It alsoincludes systems and methods for treating patients using nutritiveformulations as disclosed. In a preferred embodiment of the invention,we estimate fractional GNG in a patient, and use this to prescribe therates of parenteral and enteral energy substrate administration tosupport patient recovery. Some of the expected benefits of thistreatment are an increased healing rate and decreased hospitalizationtime. Importantly, in some patients the results of our invention maymake the difference between poor versus good recovery, and in otherpatients, the difference between life and death. The course of discoveryto use fractional GNG as a biomarker of BES and needs for energysubstrate nutrition is described here.

Inspiration for the invention arose in part from the inventors'cumulative professional experiences in public service, education, andconsultation in industry and metabolic research in exercise physiology.In particular, metabolic stresses such as the oxygen-limited conditionof high altitude, cigarette smoking, and the personal experience of oneinventor, Michael Horning (“MAH”) observing the metabolic effects of atraumatic brain injury (“TBI”) to a family member served to help theinventors conceive and reduce the invention to practice.

Note that in the current art, the term glycemia is loosely used to meanthe state of the body and blood glucose, in particular [glucose]. Butthe current invention takes the term and makes it much more precise, bydescribing underlying mechanisms of glycemic control such as GNG andfractional GNG. The term “tight” glycemic control is sometimes usedloosely in the current art to mean a more nuanced approach to control ofglucose, but is also not very precise. The current invention providessystems and methods for a sort of “exquisite” glycemic control that isvastly superior at estimating the patient's BES and meeting thepatient's nutritional needs.

In 2006, Ruthe Horning (“RH”), mother of MAH, was struck by a car whileriding her bicycle in Pacific Grove, Calif. She suffered a severetraumatic brain injury, and was rushed into emergency surgery for herfirst craniectomy. Later that night she received a second emergencycraniectomy (a bilateral craniectomy) to help reduce the swellingcreated by the subdural hematoma resulting from the injury to her brain,and her chances of survival (or good recovery) were uncertain. Over thenext several weeks of visiting RH in the intensive care unit (“ICU”) MAHmade many observations concerning the metabolic status of his motherincluding increased heart rate and temperature while she was in a coma.

From experience and training in science, MAH believed by grossobservation of RH's condition and vital signs, that his mother'smetabolic state was not that of a resting person, but rather hermetabolism was more like that of an exercising person. Coupled withthose observations MAH also noted that RH's recovery accelerated whenshe was eventually given enteral nutrition, at his urging. Thisnutritional source resulted in a marked improvement and she gainedstrength and mental faculties almost immediately.

RH was in serious need of nutritional support, but was underfed as theresult of state of the art treatment. Many months later RH regainedenough mental faculties to start to comprehend the enormity of theaccident and her injuries, and MAH made a commitment to her that hewould find a better treatment for injured patients. Thus a compellingneed was articulated that led to a path of discovery of the currentinvention. The need, in part, was for one or more key biomarkers of BES,methods for measuring these biomarkers, and nutritional treatmentmethods and formulations based on such measurements.

Over the years, the two inventors began exploring for the particularbiomarkers that could be used for BES assessment and treatment. Theinventors were looking for an ideal diagnostic method to use inevaluating metabolic state and nutritional needs, or BES, of an ill orinjured hospitalized human or animal patient. Over time, based on manystudies and empirical observation, it became clear to the inventors thatfractional GNG, that is % of glucose production from GNG, was the keybiomarker with regard to BES. While glucose Ra is somewhat useful (andusing glucose Ra and fractional GNG, one can also determine GNG rate ofproduction, since fractional GNG×glucose Ra=GNG rate of production),fractional GNG alone can be used to accurately assess the BES andnutritional needs of a patient.

While there are methods in the art that can be used to estimatefractional GNG and thus assess BES (3, 55-57) the current inventiondiscloses new, improved, simplified means to estimate fractional GNG.

The current art views a sedentary or unconscious patient as resting, andthus not needing any nutritional support beyond that of a restingpatient, because he or she is physically inactive. The current art alsomay view some critical illnesses as hypermetabolic, and in need ofadditional caloric support. Even in such cases, an accurate assessmentof BES does not exist, and the nutritional support is often delayed andthe nutritional management is based on formulas not necessarily specificto the patient's needs. Over time, the inventors realized that manypatients, including comatose, ill or injured persons, are often in acatabolic (body tissue and energy storage breakdown) state because ofthe stresses of illness or injury. Based on various empiricalobservations, the inventors have seen such ill or injured patients withfractional GNGs well above 25%, which is similar to that of stressed,intensely exercising individuals or even starving ones.

In fact, by definition, GNG involves catabolism of body tissues tosupport production of glucose, since it is a less favored, inefficientmethod of glucose production. Glucose is an essential body nutrient andunique fuel for tissues such as brain, nerves, kidneys and red bloodcells. To heal and gain strength an ill or injured person needsmacronutrient nutrition, in particular, including glucose or glucoseprecursors.

Another embodiment uses an estimate of the absolute rate of GNG inmg/kg/min, in addition to the % GNG, as a biomarker. This requires anestimate of glucose Ra, in addition to fractional GNG, in order to yieldand estimate of the absolute rate of GNG (absolute rate GNG=glucoseRa×fractional GNG).

In the late 1970's and early 1980s, using radioactive tracers of glucoseand lactate the inventors, among others (11, 23) saw that exercise andexercise training increased the ability of laboratory rats to maintainnormal blood glucose concentration (also referred to as [glucose]) byincreased capacity for making new glucose via GNG, using precursors suchas lactate. In humans and other mammals, lactate production takes placein working muscles, among other tissues, whereas the conversion oflactate to glucose generally takes place mostly in the liver andkidneys. With the advent of stable, non-radioactive isotopes, theinventors, among others, studied men resting and exercising in alaboratory at sea level and developed technology to measuregluconeogenesis in resting and exercising men. Then, in the late 1980s,the inventors, among others, were able to use stable isotopes to measureand compare glucose and lactate fluxes in men resting and exercising ina laboratory at sea level and then under the added stress of the 14,000feet elevation on Pike's Peak (9, 10). Among the many remarkablefindings of such studies were enhanced ability of the subjects to bothproduce and use lactate for energy, in part by converting lactate toglucose via GNG and directly oxidizing the remaining lactate.

In addition, using stable isotopes of glucose and lactate, theinventors, among others, (21, 22, 39) made measurements showing enhancedGNG from lactate in smokers compared to non-smokers while exercising.Subsequently, over the course of decades of experimentation theinventors became experts in the science and technology of studying humanphysiology and of using isotope tracers to measure various metaboliteflux rates in humans and other mammals engaged in exercise and otherabove-mentioned stresses.

For the inventors, lessons learned in the laboratory were complimentedby a far wider set of experiences that involved the acquisition andtransfer of knowledge that occurred within the context of generalresearch in the field. Areas of experience included providing consultingand other services to business, scientific and governmentalorganizations. Additional areas of experience included serving on reviewboards and providing editorial duties for scientific journals.

The invention benefits treatment of an ill or injured person by usingfractional GNG as the critical biomarker. While the general concept ofestimation of fractional GNG by itself is not new, see, for example (3,14, 20, 26, 27, 73, 74) the invention introduces new and improvedsystems and methods for such estimation. The invention also further usesthese estimates as a highly useful determinant of the balance of BES,e.g., catabolism vs. anabolism and nutritional needs of an ill orinjured patient in order to treat, feed and provide nutrition to thepatient appropriately. For discussion of the general concept of thegeneral use of biomarkers and measurement of various other aspects ofmetabolic flux see, for example (28, 65).

In addition to other improvements, the invention includes theimprovement of continually or dynamically estimating fractional GNG,thus providing an ongoing basis by which to understand the BES of thepatient and thus treat the patient, in addition to point measurement offractional GNG. The invention includes a new metabolic diagnostic testto assess fractional GNG to determine the underlying metabolic andnutritional status, or BES, of a patient. The scientific literatureincreasingly suggests that such measurements should be made, neitherspecifies how to specifically interpret such information in the contextof the metabolic and nutritive state of the patient, nor how to proceedon this information in terms of formulations and amounts of suchformulations (75). In underfed patients, the liver, and to a lesserextent the kidneys, are the body organs that make new glucose from GNG.The invention also includes using the information derived from the testabove to articulate information on the metabolic state and nutritiveneeds of a patient.

Estimating Fractional GNG

In a preferred embodiment, fractional GNG is estimated, which alone canbe used as a highly useful, even determinative biomarker of BES. Thebasic principle is to label a portion of the patient's body water andthen to estimate the portion of glucose production that becomes labeledvia the GNG pathway. Because a label such as deuterium can beincorporated onto different positional carbons of glucose depending onwhether the glucose was produced by GLY or GNG, such labeling can beused to estimate fractional GNG in the invention.

To function as an effective precursor label, the proportion of bodywater to be labeled must be large enough to give an accurate measurementof isotopic enrichment in both body water and blood glucose by whateverdetection mechanism is used to determine the isotopic enrichments. Itshould also be highly sensitive so that relatively small blood samplescan be taken for comfort and efficacy, and to reduce the cost ofisotopic labeling and analyses. In a preferred embodiment, that label isdeuterium, that is, the hydrogens in the water are the deuterium isotope(written as ²H or D) and D₂O is added to body water.

This deuterated water (generally commercially available at >98% purity)is generally introduced intravenously to the patient. In a preferredembodiment, an amount of deuterated water that approximates 0.3-0.5% ofbody water is given to the patient as a bolus. This amount is typicallyestimated by assuming 70% of body weight is water. Within a few hours,the labeled water both equilibrates with body water and is incorporatedinto blood glucose via the GLY and GNG pathways due to rapidisomerization between fructose-6-phosphate and glucose-6-phosphateduring the process of glucose production. The hydrogen atoms on carbon 2on glucose will be labeled via glucose production in proportion to thelabeling of body water, and can be used to validate the % labeling ofbody water number enriched with deuterium.

By contrast, hydrogen atoms on carbons 1, 3, 4, 5, and 6 of glucose willbe enriched with deuterium during transit of precursors through the GNGpathway. The hydrogen atoms on carbons 1, 3, 4, 5, and 6 of glucose willbe labeled equally during GNG due to isomerization ofglyceraldehyde-3-phosphate to dihydroxyacetone phosphate by triosephosphate isomerase and a series of equilibration reactions betweenphosphoenolpyruvate and dihydroxyacetone phosphate. The abundance oflabel at each of these carbons thus each represent GNG enrichment,though the average of these carbons (or an average of some) can be usedfor a potentially more accurate measurement. Thus, for example, thefraction of hydrogen atoms enriched by deuterium on C-1 of glucose willbe equal to that on C-5, but each will have an amount smaller than theenrichment at C-2 due to the combined pathways of GNG and GLY.

In a preferred embodiment, a blood sample is taken after this initialbolus, and the glucose is extracted using standard methods known in theart via solvents. In a preferred embodiment, the glucose is converted tothe penta-acetate molecule shown in FIG. 2, approximate MW 390, so thatthis glucose derivative can be detected in a gas chromatograph (“GC”)mass spectrometer (“MS”). The hydrogen-deuterium atoms on C-2 areremoved during ionization so that we can isolate the carbons enriched bydeuterium during GNG, thus obtaining the average GNG enrichment. Thismass to charge (m/z) ion has a non-labeled molecular mass (“m”) of 169and charge (“z”) of 1. If the ion is enriched with a deuterium at one ofthe carbons, then its m/z will be 170.

In one embodiment, the invention can be practiced withoutchromatography. Sugar measured without a chromatography step to isolateglucose from other blood sugars such as fructose and galactose wouldstill be about 95% glucose, and so a meaningful number for glucose canbe obtain without separation. Carbohydrate digestion produces a highpercentage of glucose as the fundamental energy source for cellmetabolism. Two other forms of sugars, galactose, and fructose, are alsoproducts of carbohydrate digestion. Despite the relatively highpercentage of digested fructose and galactose (about 20%), aftergastrointestinal absorption, the liver enzymes convert most of thesesugars to glucose, resulting in the 95% number.

Of course, some of the ions will be enriched at more than one carbon,and by more than one isotope. For instance, endogenous (background)isotopic enrichment of carbon in body substances by (¹³C) approximates1.09%. Similarly, the endogenous (background) deuterium enrichment isvery small, approximately 0.015%, and the target D₂O enrichment in bodywater is approximately 0.3 to 0.5%, the background deuterium will notmeaningfully affect estimation of fractional GNG as described here. Itcan in any case be corrected for, if desired.

The literature contains no reference for the method of determiningfractional GNG and isotopic enrichment in body water followingadministration of D₂O that we describe here. However, others have useddifferent methods using measurements of the isotopic enrichment ofglucose following administration of D₂O and comparing to the enrichmentin body water (20, 30, 45).

The ratio of 170/169 ions (further divided by 6, the number of hydrogenatoms on this ionized glucose fragment) divided by body water enrichmentwill thus yield an abundance value for fractional GNG. The body waterenrichment value can either be taken from the bolus to body weightapproximation described above (and, in a preferred embodiment, intendedto be 0.3 to 0.5%), or estimated from the glucose carbon 2 enrichmentvia GLY as described in this equation:

Fractional GNG=((abundance 170/abundance 169)/6)/fraction of body waterlabeled

For reference, a standard chemical representation of glucose,approximate molecular weight (“MW”) 180, in shown in FIG. 1(specifically the biologically dominant confirmation alpha-D-glucose).That same glucose molecule with each of the 7 hydrogens replaced withthe marker deuterium (D) is shown in FIG. 2.

FIG. 2 is a standard chemical representation/illustration of themolecule glucose, with all seven hydrogens replaced with deuteriums (aslabels).

FIG. 3 shows the penta-acetate glucose derivative, approximate MW 390,that is used in the GC/MS analysis method, in a preferred embodiment ofthe invention.

FIG. 4 shows a fragment of the penta-acetate glucose derivative,approximate MW 331, as well as the same molecule with different isotopes(MW 332, etc.)

FIG. 5 shows a fragment of the penta-acetate glucose derivative,approximate MW 271, as well as the same molecule with different isotopes(MW 272, etc.)

FIG. 6 shows another fragment of the penta-acetate glucose derivative,approximate MW 169, as well as the same molecule with different isotopes(MW 170, etc.)

In a preferred embodiment of the invention the body water enrichment istaken from observing the abundance of the labeled penta-acetate glucosederivative MW 331. This ion has a non-labeled molecular weight of 331and charge of 1. Since this ion retains all carbons and hydrogens of thebase glucose molecule, it is enriched by both GLY and GNG, at one of theseven hydrogens associated with the six carbons of the glucose,resulting in an ion with molecular weight of 332. Thus the abundance ofthis molecule represents enrichment by both pathways. Enrichment atcarbon 2 is by both pathways, and enrichment at the other carbons isonly by GNG.

FIG. 7 shows a schematic mass spectrum focusing on the 331 ion. Asstated, relative abundance of the 332 ion (marked with one deuterium)vs. the 331 ion represents enrichment by both GLY and GNG pathways.

FIG. 8 shows a schematic mass spectrum focusing on the 271 ion. Sincethis ion has lost the hydrogen at carbon 2, it cannot be marked by theGLY pathway. The ratio of 272 to 271 thus represents the enrichment dueonly to GNG. Since the ion may also exist in a configuration where thehydrogen is still present in the molecule, the estimation of enrichmentdue to GNG may be modified by a correction factor, in one embodiment1.0/0.9, because about 90% of the molecules exist in the configurationwithout the hydrogen at carbon 2.

FIG. 9 shows a schematic mass spectrum focusing on the 169 ion. Sincethis ion has lost the hydrogen at carbon 2, it cannot be marked by theGLY pathway. The ratio of 170 to 169 represents the enrichment due onlyto GNG. Since the ion may also exist in a configuration where thehydrogen is still present in the molecule, the estimation of enrichmentdue to GNG may be modified by a correction factor, in one embodiment1.0/0.65, because about 65% of the molecules exist in the configurationwithout the hydrogen at carbon 2.

Since we have information on both pathways, we can establish a baselinefor the amount of label produced by both the GLY and GNG pathways. Theratio of 332 to 331 represents enrichment by both pathways. When eitherthe 170/169 or 272/271 ratios (or the average of both) is subtractedfrom this, this yields an estimate of the % of body water labeled, sincebody water is the starting point for both pathways of glucose genesis.This ratio can be used to confirm the % of body water labeled, in oneembodiment. We can also have a % body water label baseline based on theamount of labeled water introduced into the body compared to body weightand/or total body water estimate (generally body water is assumed to be70% of body weight) in another embodiment. If the ratio of 332/331 minus170/169 or 272/271 or both differs from this, we can use the average orsome other combination of these numbers to establish a baseline for %body water labeled, in another embodiment.

Note that in the above schematic mass spectra, the relative abundance ofions is represented by sharp, one-dimensional lines. The abundances areessentially represented on the y-axis as intensities. In reality, theintensity readings in mass spectra show up as two-dimensional peaks(hopefully, relatively sharp). The signal/intensity/abundance of eachion is generally calculated as the area under the curve of that peak.FIG. 10 shows an actual Selected Ion Monitoring (“SIM”) GC/MS spectrumof the current invention, showing peaks for selected ions 331 and 332.In any case, it should be noted that the units of the y-axis are notgenerally important, as long as the abundance is adequate for goodchromatography, since what we care about are intensity/abundance ratios,and the ratios are dimensionless.

In a preferred embodiment, the GC/MS used is the Agilent GCMSD 5973. Ofcourse, other types of GC/MS devices or other types of massspectrometers such as liquid chromatographs can be used, provided thatthe enrichment is sufficient to be accurately detected and themolecules, or derivatives and fragments of the molecules representativeof the relevant label or labels can be detected. Other types of massspectrometers such as, but not limited to, three-dimensional quadrupoleion trap, linear quadrupole ion trap, orbitrap, sector, time-of-flight,Fourier transform ion cyclotron resonance or other detectors. Suchdetectors can be used alone or in combination (called tandem massspectroscopy), e.g., triple quadrupole, quadrupole ion trap.

The creation of ions can occur by a variety of methods and systems,including, but not limited to, electron ionization (“EI”) and chemicalionization (“CI”) used for gases and vapors, electrospray ionization,nanospray ionization, matrix-assisted laser desorption ionization(“MALDI”), inductively coupled plasma (“ICP”), glow discharge, fielddesorption, fast atom bombardment (“FAB”), thermospray,desorption/ionization on silicon (“DIOS”), direct analysis in real time(“DART”), atmospheric pressure chemical ionization (“APCI”), secondaryion mass spectrometry (“SIMS”), spark ionization and thermal ionization(“TMS”). These ionization techniques result in the transformation of themolecule to an ion or multiple fragments of ions. Variouschromatographic techniques, for example, gas chromatography (“GC”) andliquid chromatography (“LC”) can be combined with the mass spectrometerdetectors. For LC/MS the interface between liquid phase and gas phasetypically uses either nanospray ionization or electrospray ionization.

The invention can also be used with single “purpose-built” massspectrometers. Distinguished from conventional central laboratory massspectrometers, purpose-built mass spectrometers, typically are small,single biotechnology application, mass spectrometers that useminiaturized molecular traps operating near atmospheric pressure withsmall versions of pumps, ionizers, detectors and electronics needed. Ahandheld version can take a small blood sample so that tests such as %GNG can be easily and routinely sampled.

The invention can use deuterium oxide (D₂O) alone, or D₂O with eitherD₂-glucose, or [1-¹³C]glucose tracers administered intravenously uponadmission to a hospital intensive care unit (“ICU”), in preoperativepreparation or other forms of hospital admittance in order to establishbaseline values for glucose Ra, % GNG, and absolute rate of GNG. In apreferred embodiment, assuming when blood glucose is in the normal rangewithout exogenous intravenous glucose supplementation, the use of D₂Oalone would be sufficient to yield % GNG as the sole biomarker, andanalysis of % GNG alone is a preferred embodiment of the invention.

In short, estimation of % GNG consists of the intravenous administrationof tracer or tracers, a small blood sample (small enough in fact for thediagnostic to be used in infants and children), preparation of samplefor analysis and mass spectrometry to determine the mass isotopomerdistribution of the incorporation of D₂O into the product glucose andthe deuterium enrichment of the precursor body water. Our relativelyeasy, fast and cost effective invention can be easily deployed inhospitals and trauma centers throughout the world. In other preferredembodiments, we can combine % GNG estimates with other analyses (D₂Oplus either D₂-glucose (2 deuteriums at C-6), or [1-¹³C]glucose (glucosewith a carbon 13 at C-1), to yield estimates of glucose Ra and absoluterate of GNG. This would give additional information as to the todetermine metabolic and nutritional state of the patient.

Expanding on previous methods using mass spectrometry to estimatefractional GNG (20, 30, 44), we propose new stand-alone infusates anddiagnostic methods. Using oral and vascular administration of D₂O (²H₂O)and a single, small blood sample, the fractional GNG estimate anddeuterium enrichment in body water (D₂O or ²H₂O) (precursor) can besimultaneously measured using the ion fragmentation patterns resultingfrom a single mass spectrum. When the average deuterium (D or ²H)enrichment (over glucose carbons 1, 3, 4, 5, 6) is used to determine %GNG, we can call this the “averaging” fractional GNG method ofestimation.

When divided by the enrichment of deuterium in body water (i.e.,precursor), the fractional GNG estimate (glucose being the product ofthis pathway), yields the estimation of fractional glucose productionfrom GNG, which we call fractional GNG or % GNG—the terms are used inthis invention interchangeably. Instead of performing a separateanalysis for the determination of the isotopic enrichment of deuteriumin body water, we propose to use multiple fragments that result from thepenta-acetate glucose derivative and mass spectrometry. Our inventionfor the averaging GNG estimate method is to measure the total deuteriumenrichment of all hydrogens of glucose and subtracting the enrichment ofdeuterium on C-1, C-3, C-4, C-5 and C-6, the difference of which resultsin calculation of the enrichment of deuterium on C-2 of glucose. Theenrichment of deuterium on C-2 is equivalent to the enrichment ofdeuterium in body water. Expressed in another way, enrichment ofdeuterium on C-2=(1,2,3,4,5,6,6-H₇-1,3,4,5,6,6-H₆).

Therefore fractional GNG=average(1,3,4,5,6,6-H₆)/(1,2,3,4,5,6,6-H₇-1,3,4,5,6,6-H₆).

The penta-acetate derivative of glucose contains all 6 carbons and 5acetate functional groups that have replaced the native glucose hydroxylgroups. With methane chemical ionization (CI) and electron impactionization (EI) the first prominent fragments are mass-to-charge (m/z)331 and the related naturally occurring isotopomers (m/z 332, 333 and334). This “331 fragment” contains all the carbons of glucose and allthe hydrogens of the glucose molecule (i.e., C-1, C-2, C-3, C-4, C-5,C-6 and H-1, H-2, H-3, H-4, H-5, H-6, H-6 [also can be written as1,2,3,4,5,6,6-H₇] (7 hydrogens total)). The other ion fragments ofinterest in the proposed method are m/z 169 and its related naturallyoccurring isotopomers (m/z 170, 171, 172). Similar to the 331 fragment,the 169 fragment also contain all the carbons of glucose, but adifferent number of related hydrogens (i.e., C-1, C-2, C-3, C-4, C-5,C-6, and H-1, H-3, H-4, H-5, H-6, H-6 [or 1,3,4,5,6,6-H₆]). Aspects ofour invention are recognition of the loss of H-2 from the 169 fragment,and inclusion of H-2 in the 331 fragment of the penta-acetate derivativeof glucose following the administration of D₂O and the process of GNG.

Due to complete hydrogen exchange with body water during the extensiveglucose-6-phosphate

fructose-6-phosphate isomerization, the enrichment of ²H at C-2 ofglucose represents body water enrichment. Using the difference of ionintensities between the two fragments identified above yields both the“average” enrichment of deuterium using ions 169 and 170 [(170/169)/6],plus the body water enrichment calculated from the difference betweenthe M+1 ratio from ions 331 and 332 (332/331) and the M+1 ratio of ions169 and 170 (170/169). Hence, fractional GNG can be estimated bycomparing the deuterium enrichment on C-2 with the “average” enrichmentof ²H on a glucose penta-acetate derivative following the administrationof D₂O.

Alternatively stated, in one embodiment, fractional GNG can becalculated by dividing the “average” ²H glucose isotopic enrichment (theproduct) by the body water enrichment following administration of waterand D₂O (the precursor), see, for example, (20, 30, 58). Restatedanother way, ²H enrichment on C-2 of a glucose penta-acetate derivativefollowing administration of D₂O is due to both GNG and GLY. However, toreiterate, our new and novel method of measuring % GNG depends ondeterminations of the positional isomers of deuterium labeled glucoseduring GNG, an assumption that has been verified independently (20, 30).And any one or more of the GNG enriched carbons can be used to arrive atthe % GNG estimate.

To review the formulas relevant to the invention:

M+1 ratio=m/z(M+1/M); M and M+1 represent ion fragments from massspectrometry

The M+1 ratio can also be represented as (M+1/Sum(M+(M+1)). For exampleusing ion fragment 331, M+1 ratio=332/(331+332).

Mole Percent Excess (“MPE”)=M+1 ratio_(sample) −M+1 ratio_(background);sample is blood sample acquired after administration of ²H₂0, andbackground is blood sample acquired upon admittance to hospital andbefore administration of D₂O. M+1 ratio(332/331)−M+1 ratio(170/169)=body²H₂0

M+1 ratio (170/169)_(sample) −M+1 ratio(170/169)_(background)=Total MPE

Total MPE/6=average ²H enrichment of C-1, C-3, C-4, C-5, and C-6 bloodglucose penta-acetate derivative

Fractional GNG=average ²H enrichment/body ²H₂0

In a preferred embodiment, the method of invention is comprised of threeindependent parts. Part 1 comprised of two sub-parts, (1A) administeringD₂O to an ill or injured patient or to a healthy control in who GNGneeds to be measured, and (1B) measure the glucose penta-acetate ionfragmentation patterns by mass spectrometry, as described above.

Sub-Part 1A

At the start of treatment, e.g., on admission to a research laboratory,hospital or clinic for ill or injured patients, or research laboratory,for healthy subjects and scientific study, and before administration ofD₂O, a background blood sample should be drawn and prepared foranalysis. The background sample is useful because, depending on apatient's dietary and environmental history, small and variable amountsof ²H and ¹³C isotopomers naturally occur in body water, bloodmetabolites and other body compartments. After this background bloodsampling and analysis, a constant infusion of deuterium oxide willcommence (with or without a bolus depending on the whether % GNG needsto be assessed in the first several hours of admission). Because thedeuterium oxide equilibrates with the body water, a desired enrichmentcan be achieved and maintained throughout the entire hospitalizationperiod.

The desired isotopic enrichment of body water (in one embodiment about0.3-0.5%, or adequate for ion intensity comparisons from the utilizedmethod of mass spectrometry) will be adjusted by the constant infusionof D₂O and verified by the determination of deuterium enrichment on C-2(as described above using the difference in ion intensities between ionfragments 331 and 169). Alternatively the determination of body waterenrichment can be determined by isotope ratio mass spectrometry (31, 66)or using an isotopic exchange with acetone method (81). Then, whenneeded and as frequently as necessary, a small blood sample can be drawnto determine fractional GNG on an ongoing/dynamic basis. As nutritionalsupport is augmented using the following parts of the invention, thefractional GNG will be controlled, such as within the target range(20-25%) in a preferred embodiment, varied to mimic a normal dailycircadian pattern, or varied to yield any particular % GNG ranging from0 to 100% in other embodiments.

The frequency of blood sampling following D₂O administration, thesubsequent calculation of % GNG, and adjustment of enteral andparenteral nutrition is limited by the time needed for analysis. Givencurrent technology, a 2-hr frequency should be practical. However, astechnology advances, the frequency of sampling may increase. In a busyhospital setting, morning, noon and evening sampling may be necessary toestimate % GNG and other biomarkers.

Sub-Part 1B

The analysis of the data derived from the analytical process in Part 1determined from the mass isotopomer distribution of deuteriumincorporation into glucose yields the rate of GNG, which can also beunderstood as the % of Ra glucose derived from GNG precursors (mainlylactate). This is referred to as fractional GNG or % GNG. Fractional GNGis proportional to the rate of glucose production. In a normal restingperson, % of total glucose production to support the brain approximates25%, a very high percentage considering the mass of the brain incomparison to the rest of the body. Blood glucose demands increase ininjured persons regardless of the site of injury, and the balance ofglucose Ra from GLY and GNG varies depending on nutritional state, timeand metabolic needs of various body tissues. Paradoxically, followingbrain injury, cerebral glucose uptake is stunned and diminished,however, the % of cerebral glucose uptake from GNG rises, as has beenobserved by the inventors and other researchers, in studies to bepublished in the coming months.

In healthy, uninjured persons the % GNG can fluctuate between ˜10% (overfed), to ˜20-25% (appropriately nourished) to as high as ˜90% (inundernourished and catabolic patients). Our observations show that % GNGapproximates 70% for TBI persons in the ICU, as has been observed by theinventors and other researchers, in studies to be published in thecoming months.

Importantly, even though the % GNG will follow a circadian fluctuationin a traumatized or critically ill patient; this fluctuation can beminimized by the provision of nutritive support to achieve a normal(20-25) % GNG, indicative of normal metabolic and nutritive states. Bythis means, “exquisite” glycemic control and Ra glucose, without the useof conventional or intensive insulin therapy, can be achieved, which maybe of critical benefit to the patient.

In describing % GNG it needs to be understood that % GNG is a variablethat can have physiological range of 0 to 100%. Based on our work aswell as that of others, the stated target range of 20-25% in healthypost-absorptive individuals, is a biomarker for adequate nutrientdelivery in an ill or injured patient or other individual incapable oftaking adequate macronutrient nutrition, as defined by theHarris-Benedict (32) or Institute of Medicine equations (8, 51). In apreferred embodiment of the invention, nutrition is provided so that %GNG is between about 15 and 30%. In another preferred embodiment of theinvention % GNG of about 20-25% is aimed for based on studies on healthyyoung individuals 3-4 hours after having eaten (3, 24, 26, 27, 58, 73).

Part 2

Part 2 articulates the metabolic and nutritive state, also known as bodyenergy state (76) of the patient. For Part 2 the acquisition of the massisotopomer distribution of the deuterium and hydrogen content in thebody water and the glucose, taking into consideration the naturaloccurrence of isotopes of carbon and other atoms with naturallyoccurring isotopes, consists of selective ion monitoring (“SIM”) of themass to charge ratio (m/z) of the ions of interests coupled with anintegration of the SIM to deduce the response factor associated with theabundance of the ions of interests as they relate to the precursor andproduct relationship.

The invention also includes a method and system to compare the precursorand product relationship based on the patient's baseline measurement andthe daily (or multiple daily) measurements taken from the patient. Theinvention, in a preferred embodiment, has a database that informs thebasis from which the nutritional status was evaluated and theprescription of nutritional support was determined. With the additionalprocess of part 1, the database will contain variables for the relevantions (e.g., 331, 332, 272, 271, 169, 170) to calculate fractional GNGand body water enrichment as it relates to infusion of the tracerdeuterium oxide and the precise, prescription nutritional support. Aswell, the system will contain non-identifiable data on patients, theseverity of injury on entry into the study, the initial % GNG, theenteral and parenteral nutrition provided, and patient outcomes.

It will be appreciated that many of the described methods can beintermediated and implemented automatically by a computer, orspecial-purpose hardware, or some combination of both, as such systemsare well known in the art. Specialized software or hardware of theinvention could read the intensity of signals provide my mass spectraand automatically calculate the ratios and other important data to givea reading of fractional GNG. Such readings could automatically be storedin databases or computer memory and presented to users in various visualforms. The software could also make recommendations as to feedingprotocols and times, frequency of patient sampling, or simply carry outthese methods automatically.

The invention as such can be implemented on any suitable computersystem. A typical, general purpose computer system suitable forimplementing the present invention includes any number of processorsthat are coupled to memory devices including primary storage devicessuch as a read only memory, random access memory and hard drives. Anyone of many data and database architectures can be used to store andretrieve methods, protocols and recommendation, to store data, and tocommunicate with server side assistance through the Internet and othernetworks.

A hardware system may be specially constructed for the requiredpurposes, or it may be a general-purpose computer, such as a servercomputer or a mainframe computer, selectively activated or configured bya computer program stored in the computer. The processes presented aboveare not inherently related to any particular computer or other computingapparatus. In particular, various general-purpose computers may be usedwith programs written in accordance with the teachings herein, or,alternatively, it may be more convenient to construct a more specializedcomputer system to perform the required operations.

Such a general-purpose computer system suitable for carrying out theprocessing in accordance with one embodiment of the present inventioncan be a server computer, a client computer, or a mainframe computer.Other computer system architectures and configurations can be used, madeup of various subsystems described below, includes one or moremicroprocessors (or central processing units). Using instructionsretrieved from memory, the microprocessor controls the reception andmanipulation of input data, and the output and display of data on outputdevices.

Part 3

Part 3 relates to nutritive methods, formulations and amounts. With theprescription of medicine determined following parts 1 and 2 dictated bythe invention for each patient, the attending physician shall administerthe level of nutritive support to administer to each patient tonormalize the % GNG. Further, the attending physician or other healthcare professional shall utilize information from continualdeterminations of GNG and nutrient delivery following application ofparts 1 and 2 of the invention for each patient as they recover or asconditions change. The invention will prescribe feeding protocols forthe patient, as follows, based on general nutritional concepts describedin (8, 51).

(A) A preferred form of treatment will consist of the intravenousinfusion of the gluconeogenic precursor, including any of the followingin combination or alone: L-(+)-lactate salts, other lactate compounds,L-(+)-pyruvate salts, other pyruvate compounds, L-(+)-lactate alone,lactate plus other amendments included in. Lactate, pyruvate and similarnutritional molecules are herein referred to as monocarboxylatecompounds (“MCC”). The metabolic precursors of glucose in the GNGpathway are herein called GNG precursors. These include many MCCs, suchas those listed herein, as well as other compounds, such as some aminoacids (e.g., alanine) and glycerol compounds.

These nutritive formulations are referred herein as cocktails,infusions, formulations, MCC cocktails and GNG precursor cocktails. Toprovide nutritive support and reverse body catabolism to anabolismfollowing trauma or chronic illness, the rate of MCC infusion wouldrange from high (13) to low (13) as governed by the individuallymeasured % GNG and glucose appearance rates:

High (Maximum) MCC Cocktail infusion rate (mg/min)=glucose Ra(mg/min)  A1

Low (Minimum) MCC Cocktail infusion rate (mg/min)=glucose Ra (mg/min)×(%GNG)=absolute rate of GNG  A2

In its simplest form, the MCC cocktail would be sodium-L-(+)-Lactateprepared by titrating L-(+)-Lactic acid with NaOH (24, 52, 55-57).Briefly, the MCC infusion cocktail is prepared by mixing 30%L-(+)-lactic acid solution (e.g., Sigma) in 2 N NaOH to pH 4.8. Theinvention, in one embodiment, specifies an initial infusion rate woulddeliver 11-50 (micro Moles per kg of body weight per minute)μMoles/kg/min, with maintenance infusion rate targeting blood lactateconcentration of 3.5-4.5 mM. Higher blood lactate levels (6 mM) havebeen seen without ill effects (57, 71). Consistent with section methodsdescribed above, assuming a formula weight of 112 mg/mMol for sodiumlactate, an infusion rate of 11 μMoles/kg/min would deliver the massequivalent of 1.0 mg/kg/min of glucose, an infusion rate of 23μMoles/kg/min of sodium lactate MCC would deliver the mass equivalent of3 mg/kg/min glucose, whereas an infusion rate of 50 μMoles/kg/min woulddeliver the mass equivalent of 4.5 mg/kg/min of glucose. Ideally, theMCC is prepared from highest purity materials, is pathogen free,certified for human pharmaceutical use and is delivered into a largecentral vein, but peripheral vein can be used if administered withphysiological saline to minimize osmolality and pH effects at theinfusion site that might provoke phlebitis of hemolysis.

In one embodiment, the starting MCC infusion rate is approximately 3mg/kg/min, or in an alternate embodiment, 100% of glucose Ra, as hasbeen observed by the inventors and other researchers, in studies to bepublished in the coming months. In one embodiment, this can be done evenwithout estimating % GNG, Ra glucose or other biomarkers. This route andamount of vascular lactate administration has been shown to be safe(55-57, 71). The amount also corresponds to the average empiricallydetermined Ra glucose in TBI patients (74), as has been observed by theinventors and other researchers, in studies to be published in thecoming months.

The invention provides for adjusting the nutrition, including MCCinfusion rates, such that a target % GNG is achieved. To some, resultsof studies of the extent of gluconeogenesis in humans might seem quitevariable; however, if results are viewed from the context of subjecttime since last eating, then a clear pattern emerges: GNG is suppressedas nutrients enter the gut, portal and circulation (e.g., 0-15% ofglucose Ra), and 20-25% of glucose Ra 3-4 hr after a mixed, CHO(carbohydrate) containing meal, and the percentages rises continuouslythereafter (74).

(B) An alternative and also preferred form of treatment will involveProcedure A plus either (B1) enteral nutrition via nasal gastric ornasal jejunal tube, or parenteral nutrition (B2) via intravenouscatheter. If these, Method B1 is useful because nutrients will enter thestomach and reach intestines, portal circulation and liver, therebyeliciting physiologically appropriate and anabolic local intestinal andlong neural endocrine reflexes as well as general endocrine responseswith signals reaching the liver, pancreas, muscles, heart, adipose, andbrain including hypothalamus regarding the presence of appropriatenutritive energy support (79).

(B1a) The clinician shall proceed as in (A), above, but, as well,provide enteral nutritive support according to various protocols, suchas the Appropriate Macronutrient Distribution Ranges (“AMDR”) as definedby the Institute of Medicine (“TOM”). These AMDR ranges are:carbohydrate in the range of 45-65%, protein in the range of 10-35% andfat in the range of 20-35%, with total daily energy input as determinedby the TOM equations for men and women (8, 51) assuming no physicalactivity, but a 10% increase in total daily energy expenditure (“TEE”)to support hyper-metabolism of trauma and cover energy needs for tissuerepair such that physical activity level (“PAL”) (8, 51)=1.1:

Men: TEE=1864−9.72×age [yr]+PAL×(14.2×weight [kg]+503×height [m])

Women: TEE=1387−7.31×age [yr]+PAL×(10.9×weight [kg]+660.7×height [m])

(B1b) The clinician shall proceed as in (A), but as well provide enteralnutritive support from various protocols, such as according to theAppropriate Macronutrient Distribution Range (“AMDR”s) as defined by theInstitute of Medicine (51), and that energy is delivered according tothe Harris-Benedict Equations (32):

Men: TEE=66.473+13.7516 W [kg]+5.0033 H [cm]−6.7550 Age [yr]

Women: TEE=655.0955+9.5634 W [kg]+1.8496 H [cm]−4.6756 Age [yr].

(B2) The clinician shall proceed as in (B), but nutrients will beadministered into the blood via an indwelling catheter. Monitoring andadjustment of MCC and macronutrient infusion rates will be similar to(A) and (B1).

(C) Another embodiment of the invention relies on robust, scientificallydetermined underpinnings of the metabolic and GNG responses to trauma,including neurotrauma, as has been observed by the inventors and otherresearchers, in studies to be published in the coming months, and is tobe regarded as an emergency procedure when isotopes and analyticalequipment are unavailable and the patient needs to be sustained untilrelocation to an appropriately equipped facility.

(C1a) The clinician shall commence intravascular infusion ofNa⁺-L-(+)-Lactate at the rate of 3 mg/kg/min plus enteral nutritivesupport according to the AMDRs and TEE estimates as given by theInstitute of Medicine (“10M”) (8, 51).

(C1b) The clinician shall commence intravascular infusion ofNa⁺-L-(+)-Lactate at the rate of 3 mg/kg/min plus enteral nutritivesupport according to the AMDRs and TEE estimates as given by Harris andBenedict (32).

(C1c) The clinician shall commence intravascular infusion ofNa⁺-L-(+)-Lactate at the rate of 3 mg/kg/min plus parenteral(intravascular) nutritive support according to the AMDRs and TEEestimates as given by the TOM (8, 51).

(C1d) The clinician shall commence intravascular infusion ofNa⁺-L-(+)-Lactate at the rate of 3 mg/kg/min plus parenteral(intravascular) nutritive support according to the AMDRs and TEEestimates as given by the Harris-Benedict equations (32).

(D) Another embodiment of the invention also relies on robust,scientifically determined underpinnings of the metabolic and GNGresponses to trauma and is to be regarded as an emergency procedure whenisotopes, analytical equipment and MCCs are unavailable and the patientneeds to be sustained until relocation to an appropriately equippedfacility. Such locations could include, for example, a battlefield or arural setting.

(D1a) In a facility unequipped to determine % GNG the clinician shallcommence intravascular infusion of D-Glucose at the rate of 1-2mg/kg/min and enteral nutritive support according to the AMDRs and TEEestimates as given by the IOM (8, 51). The inventors have seen a rate of3 mg/kg/min in TBI patients, as has been observed by the inventors andother researchers, in studies to be published in the coming months.However, experience teaches that it is difficult to maintain thedesirable [glucose] in severely injured patients (78). In severelyinjured patients giving exogenous glucose (usually in the form ofdextrose) may induce hyperglycemia thereby eliciting an insulin responseor the need to administer insulin due to undesirably high [glucose].

This undesirable process can be described as a pattern of clinicians toclumsily trying to ride a “metabolic roller coaster” of intravenousdextrose followed by insulin and yet again dextrose, and the currentinvention can eliminate this problem. Importantly, while spending time,effort, resources and attention by managing [glucose] in patients byalternately adjusting infusion rates of dextrose and insulin, theclinician obtains little useful information or meeting patient nutrientneeds. The current invention will enable clinicians to meet patientnutrient needs and serves to save clinician time and effort and releasesclinicians from the responsibilities associated with managing theaforementioned metabolic roller coaster.

(D1b) The clinician shall commence intravascular infusion of D-Glucoseat the rate of 1-2 mg/kg/min and enteral nutritive support according tothe AMDRs and TEE estimates as given by Harris and Benedict (32).

(D1c) The clinician shall commence intravascular infusion of D-Glucoseat the rate of 1-2 mg/kg/min and parenteral (intravascular) nutritivesupport according to the AMDRs and TEE estimates as given by the IOM (8,51).

(D1d) The clinician shall commence intravascular infusion of D-Glucoseat the rate of 3 mg/kg/min and parenteral (intravascular) nutritivesupport according to the AMDRs and TEE estimates as given by theHarris-Benedict equations (32).

(D1e) If enteral or parenteral support is unavailable, the clinicianshall commence intravascular infusion of D-Glucose at the rate of 3mg/kg/min which is the empirically derived best estimate of body glucoseflux following a TBI or other injury, illness or situation, as has beenobserved by the inventors and other researchers, in studies to bepublished in the coming months. This elevated value of glucose flux thatoccurs after TBI or other injury, illness or situation, is indicative ofa “hypermetabolic” state would be in contrast to the depression inglucose flux as might occur in a “hypometabolic” state, such as advancedageing (see below, vide infra).

In a preferred embodiment, nutritive support treatment targets are % GNG20-25%. In another preferred embodiment plasma [glucose] is targeted as5-7 mM. In another preferred embodiment, plasma [lactate] is targeted as3-4 mM. These targets can be achieved by adjusting MCC, enteral andparenteral administration rates either singularly, or in combination.However in methods C and D, % GNG will not be known. In these cases, inaddition to adjusting MCC, enteral and parenteral administration rates,insulin therapy may be indicated above a certain [glucose] such as 7.8,or below 5.6 mM (75).

The robust nature of the GNG response to the stresses of injury can besupported by provision of an MCC, that can be comprised of one, orcombinations of the following: sodium L-(+)-lactate, arginyl lactate,glycerol, glycerol tri-lactate, sodium L-(+)-pyruvate, arginyl pyruvate,glycerol tri-pyruvate, glycerol tri-acetate, β-OH-butyrate oracetoacetate [in which all monocarboxylate enantiomers areL-(+)-enantiomers], or mixtures thereof in which the relative amount ofany single constituent could range from 0-100%. As well, embodiments ofa MCC cocktail could include Ca⁺⁺, Mg⁺⁺, and K⁺-salts of lactate,pyruvate, alanine, β-OH-butyrate, acetoacetate, etc., as all are saltsof monocarboxylic acids. However, sodium ion (Na+) is the main cation inplasma, normally 145 mM, other cations are far less abundant in plasma.For instance, normal values for K⁺, Ca⁺⁺, and Mg⁺⁺ are, respectively, 4,2.5 and 1.5 mM. Hence, a mixture of inorganic lactate salts comprised ofNa⁺, K⁺, Ca⁺⁺, and Mg⁺⁺ would be given in the ratio of 145, 4, 2.5, and1.5. In this embodiment of invention, the main anion would be lactate,but phosphates (PO₄ ³⁻), hydrogen phosphate (HPO₄ ²⁻) and dihydrogenphosphate (H₂PO₄—), in the amount of 1.0 mEq would be provided as well.Because Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, and H₂PO₄— are present in the plasma ofhealthy individuals at levels of 145, 4, 2.5, 1.5, and 1.0milliequivalent per liter (mEq/l, this particular embodiment of MCCcould be termed “Sanguisal” from the Latin words for blood (sanguis) andsalt (53). The provision of sodium and other cations as a means todeliver lactate anions in an MCC has the advantage of reducing brainswelling following TBI, as has been observed by the inventors and otherresearchers, in studies to be published in the coming months and (15) aswell as providing nutritive support to intensive care patients followingtrauma (70).

As an alternative to Sanguisal-L that uses lactate as the major anion(vide supra), Sanguisal-P will involve the use of pyruvate (P) as themajor anion, while at the same time maintaining the above-stated levelsof cations {Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, and H₂PO₄— as are present in the plasmaof healthy individuals: 145, 4, 2.5, 1.5, and 1.0 mEq/l}. Like it'scounterpart lactate, pyruvate is the precursor to lactate in glycolysis.Further, pyruvate is an oxidizable fuel and GNG precursor whosemetabolism may affect cell redox status if converted to lactate.Although typically present in 1/10 or lesser concentration compared tolactate, pyruvate has been introduced into the systemic circulation oflarge mammals in which the circulation has been interrupted to mimiccardiac arrest (61-63, 68). In such cases 100% pyruvate infusion raisesthe circulating pyruvate level, but more so, the circulating lactatelevel achieving a circulating L/P of 2-3 (69). None the less, because ofits chemical structure, pyruvate has the advantage over lactate ofserving as an antioxidant in the myocardium subjected to reperfusioninjury, and, by extension exogenous pyruvate my also serve to scavengefree radicals in the brain after blood flow is interrupted.

A problem with regard to the use of Sanguisal-P, in contrast to the -Lform, is that in circulation pyruvate is rapidly converted to lactatedue to the effects of lactate dehydrogenase in red blood cells and thelung parenchyma (41). Hence, for the antioxidant properties of pyruvate(48), the optimal site of Sanguisal-P infusion would be in the carotidartery or ascending aorta. Still, by the ability to infuse mixtures ofSanguisal-L and Sanguisal-P into the systemic circulation, ascending orcarotid artery, the clinician would have the opportunity to affect redoxstatus in an injured brain. However, because carotid or ascendingcatheterization is uncommon even in the ICU, systemic solutions of avery low L/P ratio (i.e., high P/L) such as infusion of 100%Sanguisal-P, or a mixture of Sanguisal-P/Sanguisal-L of 2.0 (L/P=½ inthe infusate), would serve to raise the arterial level of pyruvaterelative to that of lactate.

It should be noted that one issue with aqueous pyruvate-containingsolutions is the spontaneous degradation of pyruvate and theaccumulation of unintended and possibly toxic substances in aqueousconditions. Accordingly, the manufacturing and storing Sanguisal P andSanguisal L-P mixtures needs to be anhydrous. Water (pure and sterile)can then be added to he formulation immediately before delivery to thepatient.

In using Sanguisal-L, Sanguisal-P, or other sodium salts of lactate orpyruvate the assumption is that the patient to be supported has normalrenal function and is capable of managing the sodium load. In the eventthat the patient does suffer renal failure, then the use of Sanguisal orother sodium-based salts will need to be reduced to the levels wherenormal sodium, potassium and other plasma cations are in the normalrange. In this case also, substrate availability in patients can bemaintained by combinations of intravenous Dextrose (a synonym forglucose), with and without insulin, and enteral nutrition as describedin section D, above. Still, however, % GNG will serve as the biomarkeraround which to provide enteral and parenteral nutrition.

Alternative Methods to Estimate % GNG

The invention is comprised of a three-part process to assess themetabolic status and deliver macronutrient energy to an ill or injuredhuman or other mammal. The first part is to estimate the % GNG, thefavored method is to utilize deuterium oxide (D₂O) alone, or D₂O witheither D₂-glucose, or [1-¹³C]glucose tracers administered intravenouslyupon admission to a hospital ICU (20).

One advantage of the new method is that the tracer needs to be givenonce that will suffice for measurements of GNG to be made daily forseveral days and a constant infusion can be started to offset thedilution of the enrichment of the deuterium oxide caused by intake offluids. However, we have used primed-continuous infusions to measureglucose recycling or lactate incorporation lactate into glucose. Theformer method (glucose recycling) requires using D₂-glucose (that doesnot involve carbon recycling, and a ¹³C-glucose tracer (e.g.,[1-¹³C]glucose) in which the carbon recycles (26, 27). In thiscarbon-recycling method, % GNG=(100) Ra Glucose (from ¹³C-glucose)-RaGlucose (from D₂-glucose)/Ra Glucose (from D₂-Glucose). Disadvantages ofthis method are that tracers will need to be given continuously overdays, perhaps leading to weeks, and that assumptions need to be madeover the extent of carbon isotope dilution in the Krebs (tricarboxylicacid) Cycle (37, 38).

Another method also uses deuterium oxide and measures the deuteriumincorporation on glucose carbons using the mass isotopomer analysistechnique with the aldonitrile penta-acetate andmethyloxime-trimethylsilyl derivatives (42).

Another alternative method also uses deuterium oxide but measures theenrichment at carbon 2, 5, and 6 of glucose using the HMT(hexamethylebetetramine) derivative. Disadvantages of this method arethe complexity in preparing the derivative (44, 45).

Another alternative method involves primed-continuous infusion ofD2-glucose (and a ¹³C-lactate tracer (e.g., [1-¹³C]lactate) (3) in whichthe carbon from lactate recycles to glucose. Disadvantages of thismethod are that tracers will need to be given continuously over days,perhaps leading to weeks, and that assumptions need to be made over theextent of carbon isotope dilution in the TCA Cycle.

Another alternative method involves the primed-continuous infusion ofH¹³CO₃— (i.e., ¹³C-bicarbonate) that will be fixed as the result ofisotopic equilibration during the process of gluconeogenesis (37, 65).Disadvantages of this method are the tracer needs to be givencontinuously over days, perhaps leading to weeks. Again, the methodrelies on assumptions on the extent of carbon exchange during GNG, withthe extent of isotopic dilution subject to metabolic state of theindividual (38).

Another alternative method involves the use of mass isotopomerdistribution analysis (MIDA) using a labeled glycerol precursor.Disadvantages of this method include the large amount of tracer neededto make the measurement. (35, 58).

Another alternative method involves the use of [U-¹³C]glucose (72). Thismethod was enhanced to the “Reciprocal pool model” by (34).Disadvantages of these methods are that the tracers need to be givenover days and potentially weeks.

Still other methods for assessing GNG in vivo do not involve isotopomerdetection via mass spectrometry, but instead rely on nuclear resonancespectrometry (MRS, or NMR), e.g., (46). The literature on isotopomerdetection via MRS is less extensive, but the method may prove to be moresensitive, for instance extending the time between frequency of dosingwith D₂O to estimate GNG, or % GNG. However, the large amount ofdeuterium oxide needed for the measurement may lack efficacy.

In a preferred embodiment to measure fractional GNG, D₂O, also calleddeuterium oxide or heavy water, either alone or in combination with[6,6-²H]glucose (i.e., DD-glucose or D₂-glucose) or [1-¹³C]glucose)(20), is administered intravenously. Then, after a set amount of time ablood sample can be obtained, and the mass isotopomer distribution inthe blood sugar glucose produced from the heavy water precursor can beanalyzed to determine the percentage contribution of gluconeogenesis (%GNG) to the total rate of hepatic plus renal glucose production,alternatively termed glucose appearance (Ra glucose) that can bedetermined from the isotopic enrichment (“IE”) of D₂-glucose in blood.Beyond the isotope tracer method noted by example for measurement of %GNG, other methods also exist to determine % GNG, see below (3, 26).

The major GNG precursor is the monocarboxylate, 2-hydroxy-propionate(also known as lactate) (3, 40, 54), which is also a major energysubstrate for most body tissues (5-7), including the brain (28, 33, 64).As well, to lesser extents, the salts of other monocarboxylic acids arealso gluconeogenic precursors; these include: pyruvate, acetate,acetoacetate, β-hydroxybutyrate, and related compounds, see below.Because at physiological blood pH (about 7.4) monocarboxylic acids(lactic, pyruvic, acetic, β-hydroxybutyric, acetoacetic acids, etc.)will be dissociated to protons and respective anions, here we refer themonocarboxylate anions lactate, pyruvate, acetate, acetoacetate, andβ-hydroxybutyrate.

According to the invention, in one embodiment, the fractional GNG isused to determine nutritive support rate to achieve preferred glucose Raand fractional GNG. For example, an injured patient may have anincreased metabolic rate (hypermetabolic) and thus need both glucose andGNG precursor support. By contrast, an aging or chronically ill patientmay have a depressed whole body metabolic rate (hypometabolic), butstill an elevated need for GNG precursors without glucose support. Evenwith knowledge of [glucose], which is commonly measured, but without aclear picture of the fractional GNG rate (our biomarker for themetabolic and nutritive state of the patient), a clinician may notrecognize that the patient is in a catabolic state and is degradingessential body stores to provide the precursors and energy for GNG.

Conversely, a clinician might induce a nutritional state that rendersthe patient overfed by administering too much nutrition because a lackof knowledge of the underlying metabolic and nutritional status of theindividual patient. An overfed patient can result in significantmetabolic stress and may result in complications including prolongedmechanical ventilation, infection risk, delayed hospital discharge andeven increased morbidity (29, 47). Indeed, without knowledge of thefractional GNG and based on false assumptions of previous art, aclinician may be unaware of the body's attempt to supply glucose fromgluconeogenesis, and, in fact, the clinician may inadvertently act tosuppress GNG (78).

Application of the Invention to Various Injuries and Illnesses

By way of describing importance of our method for providing nutritivesupport to injured patients is to describe the condition of TBI (alsoknown as intracranial injury). Such injuries occur when an externalforce suddenly impacts, and causes injuries to the brain (15). Often themechanism of injury produces two, or more lesions, one a laceration orcontusion at the site of impact or cranial penetration, and the other acontralateral contusion injury if the force of impact is sufficient toaccelerate the brain such that it forcibly contacts the cranium at avector directed by the initial impact. TBI can be classified based onseverity, mechanism (closed or penetrating head injury), or otherfeatures, e.g., occurring in a specific location or over a widespreadarea (15, 28). Head injury usually refers to TBI, but is a broadercategory because it can involve damage to structures other than thebrain, such as the scalp and skull (15). TBI is a major cause of deathand disability worldwide, especially in children and young adults andthe elderly. Causes include falls, vehicle accidents, and violence.

Typically, following severe TBI, patients are unconscious in a coma, areon life support in a hospital intensive care unit (“ICU”), and have lessthan 100% chance of surviving and less than 100% chance of regainingpre-injury cerebral functions if they do survive (28). More and more,sports related concussions in American football and other activities,involving loss of consciousness, or other neurological symptoms such asdizziness, nausea, the patient “seeing stars” and behavioral changes arebecoming recognized as mild forms of TBI. Especially of concern isgrowing recognition of the frequency and severity of sports-relatedconcussions to student athletes, still undergoing neural development,and who are particularly sensitive to repeated cerebral injuries. Andfinally, another source of TBI occurs to soldiers concussed, and injuredin the field as the result of improvised, and other explosive devices.

Given this unfortunate background of TBI following vehicle accidents,falls, violence, sports and warfare, our method of determining the rateof GNG following trauma and restoring the rate to normative levels isbecoming increasingly important because of the frequency and severitiesof injuries.

The description includes examples of patient treatment following traumaand chronic illness. The invention includes, methods for managing thoseextreme and other cases and instances when assessment of BES, diagnosisand treatment are appropriate. Such examples include, but are notlimited to assessing BES of patients or others before and after surgery,before and after drug treatment or dietary intervention, or anysituation in which knowing, or standardizing BES is essential fordetermining outcome of any treatment, or establishing the effect of saidtreatment on humans or other mammals.

GNG is part of normal physiology that includes the “fight-and-flight”response to emergency situations that is well-known in the art, see forexample the classic work of Selye (67), and the reference books byCannon, W. B. “The Wisdom of the Body” (17) and Brooks (12)). In bothhealthy and uninjured persons, gluconeogenesis works to maintain bloodglucose concentration, or glycemia, in the normal range in the earlymorning hours after the previous evening's meal has been digested andnutrients cleared from the blood, and when the maintenance of glycemiadepends on GNG (54). Commonly stated examples given to illustrateimportance of the fight and flight response are evolutionary in nature,such as the need to flee predators, or catch large game animals.

While interesting, such examples are remote from contemporary humanexperience, but injuries and illnesses persist in society and areassociated with comorbidities, including nutrient deprivation. Seen inthe context of contemporary fight and flight responses, following TBIand other forms of trauma of the critically ill or injured patient theneed to provide blood glucose is paramount because glucose is anessential fuel for the brain, as has been observed by the inventors andother researchers, in studies to be published in the coming months, andother tissues such as nerves, red blood cells and the kidneys (8, 51).Indeed, the dietary reference intake (“DRI”) for total dailycarbohydrate (“CHO”) consumption was established based on the glucoseneeds for the brain (8, 51). Therefore, not only is the total body needsfor glucose increased following TBI, but also elevated is the rate ofgluconeogenesis from lactate and other GNG precursors, as has beenobserved by the inventors and other researchers, in studies to bepublished in the coming months.

The body's need for GNG is increased following TBI, and mimics thestarvation state in which body tissues are cannibalized (catabolized ordegraded), as is well-known in the art, for example see the textbook byBrooks (12). This pathway supplies byproducts of carbohydrate (e.g.,lactate and pyruvate), amino acids (e.g., alanine), and fats (e.g.,glycerol and ketones such as β-hydroxy butyrate and acetoacetate) (16).Of these, lactate is by far the most important GNG precursor, easilyseen during exercise when blood lactate concentration is elevated (3-5,7). Crucially, even though GNG provides a short term, fight-and-flightresponse to the emergency need for a glucose supply to the brain, nervesand other glucose requiring tissues, the price of GNG is to sacrifice(catabolize) essential body substances and tissues. Crucial also is therealization that the basic needs for glucose and other macronutrients byglucose-requiring tissues continue unabated whether those tissues aretraumatized, or not. Hence, key underlying concepts of our invention arethat glucose and other macronutrients are required, always, that therequirement is the same as normal or increased after trauma, and thatthe rate of GNG is a critical marker of body tissue catabolism followinginjury, and alone can be used to direct nutritional treatment.

Derived from such knowledge gained through innovative technology areclinical procedures and materials to support glucose Ra while minimizing% GNG, because GNG is the best real-time measure of body wasting. Aswell, we assert that these methods have application beyond TBI to thetreatment of trauma and chronic and infectious illnesses, in general,because the same methods support the body corpus overall that iscomprised of injured and non-injured tissues all requiring nutrientsupply.

For a traumatized patient, cachexia, or body-wasting syndrome, is aproblem because basal metabolic rate is elevated, but the person isunable to eat. Body weight loss, muscle atrophy and weakness and fatigueresult leaving a person diminished even if they survive. In addition totrauma, other conditions and illnesses of life are accompanied by lossof appetite and cachexia. These include aging, infectious diseases(e.g., tuberculosis and AIDS), chronic diseases (chronic obstructivelung disease, multiple sclerosis and congestive hear failure), cancerand some autoimmune disorders. Regrettably also, exposure toenvironmental toxins (e.g., mercury) can result in cachexia, andoccasionally there are some who are unable to access food and arestarved. All such sick individuals and chronically ill patients who areunable to ingest adequate macronutrients to maintain body requirements,let alone restore health would also benefit from treatment by theinvention we describe.

It has long been recognized that patient care in the ICU necessitatesthe monitoring of blood glucose concentrations due to the stress fromcritical illness. Some have identified insulin resistance andunsuppressed gluconeogenesis (i.e., when negative feedback from risingblood [glucose] fails to down regulate GNG) that results in the requiredmonitoring of blood glucose in over 90% of ICU patients (83).Additionally, various attempts to control blood glucose concentrationincluding hyperglycemia with blood glucose concentration of 180-215mg/dL is considered beneficial.

Conversely, others have attempted to regulate blood glucoseconcentration to values between 80 and 110 mg/dL in an attempt tomaintain a strict normal glycemic range. However, because the balance ofglucose production and removal are unknown, and not considered in thesimple measurement of blood glucose concentration, using blood glucoseconcentration as the diagnostic and insulin as the treatment often leadsto wide swings in blood glucose concentration. Regrettably, despitediligent efforts on the part of clinicians attempting to maintainpatients by dextrose drip and pushing insulin to normalize blood glucoseconcentration, the patient will be maintained at the perceived idealblood glucose concentration range for only short periods of time.Additionally, the risk of hypoglycemia from too aggressive insulintherapy is also a serious risk and increased with intensive insulintherapy. Recognizing the difficulty of using dextrose and insulintherapy to maintain blood glucose concentration in injured and illpatients others have created computer-assisted record keeping devices toassist in therapy (59), while others have studied the benefits andliabilities of early versus late parenteral complement to enteral andoral nutritional support to achieve better patient outcomes and shorterhospitalization times (18). The methods fail for lack or knowing theunderlying mechanisms of flux (metabolite appearance and disposal).

A better understanding of the pathophysiology resulting from the stressof critical illness is desired and, in fact, needed to better care forpatients. Now, by means of our invention, using fractional GNGmeasurements a clinician shall be able to determine the exactnutritional and metabolic state of the patient, and therefore, be ableto effectively intervene in illnesses such as, but not limited to,preterm infant care, complications from pregnancy requiringhospitalization, pre-operative surgery preparation and post-surgerymonitoring, stroke, aneurism, terminal illness, urinary sepsis, cardiacfailure (post-cardiac surgery), esophagectomy for carcinoma,subarachnoid hemorrhage, ileus, subdural hematoma, pulmonary sepsis,cardiac failure (post-myocardial infarction), respiratory failure(chronic obstructive pulmonary disease), oropharyngeal abscess, coronarybypass, resection of thoracic aneurysm, starvation, burns, severe acuterespiratory syndrome (“SARS”) and potential epidemics and pandemicsrelating to influenza can be treated more precisely according to theindividual nutritional needs based on the patients energy demands fromtheir individual metabolic state.

The term “patient” often refers to an individual (human, other mammal oreven other animals) suffering from injury or chronic disease. It canalso refer to acutely or chronically stressed individuals such aspremature infants, the chronically malnourished, underfed and physicallyexhausted athletes, soldiers and manual laborers, subjects in studies ofpharmaceutical development, and many others. It can and here is intendedto apply to individuals who also do not fit obvious or conventionalnotions of injured or ill patients, but can benefit from diagnostic,feeding or other treatments.

In fact, fractional GNG has already been measured in persons sufferingmany of the illnesses listed above; however, to date none haverecognized that the nutritional needs of ill and injured patients couldbe met by targeting a constant range. The inventors, among others, haveobserved that a 12-hour fasts results in a fractional GNG of about 40%,while starvation over several days will drive fractional GNG above 90%.According to the invention, treatment of an ill or injured individualwould involve negative feedback control of enteral nutrition and MCCinfusion: response to high % GNG would be increasing enteral nutritionand MCC infusion, whereas low % GNG would mean over nutrition and theneed to reduce feeding rate. A range of near 25% is generally anappropriately fed state, 3-4 hr after a balanced, CHO-containing meal.In healthy, uninjured individuals, a low level of fractional GNG, around10% would be measured soon after they consumed a balanced,CHO-containing meal. A low fractional GNG of around 10% in a comatoseindividual, such as a TBI patient in an ICU, a GNG of 10% is unlikely,as has been observed by the inventors and other researchers, in studiesto be published in the coming months, but would be indicative of overfeeding and the need to reduce enteral feeding and MCC infusion until %GNG is in the 20-25% range, thus avoiding some of the consequences asdescribed above. More typical of a TBI patient in the ICU would be %GNG >40%, thus requiring increased provision of enteral nutrition andvascular MCC infusion. In the case of a brain-injured person, theapproach should be to maintain glycemia with both enteral nutrition andMCC infusion, the latter being important to supply cerebral nutritionand electrolytes to reduce cerebral swelling and minimizinghyperglycemia from dextrose.

In one embodiment, fractional GNG of 20-25% is aimed for, in another15-35%. Regrettably, none of the current art has recognized thatfractional GNG is the key biomarker of the pathophysiology related tocritical illness. Regrettably also, none have used knowledge offractional GNG to assess patient nutrient needs. We have devised suchmethods for intravenous, oral or gastric nutrient delivery to patients.

Large and extensive studies have been conducted to understand thebenefits associated with nutritional support (18). Such studies havegone to some lengths to understand the patient's nutritional needs, butthey have not found a good biomarker to indicate if patients are wellnourished, overfed or underfed. Without the underlying metabolic stateof the patient it is impossible to know what the exact nutritional needsare of that patient. Therefore, despite studying over 5000 patients, theabove study was unable to determine a clear advantage to earlyparenteral nutrition to complete the enteral nutrition. They did,however identify negative consequences from overfeeding andunderfeeding, but it is not clear that they are able to ascertain theprecise nutritional status of their patents.

Some studies (65) have used indirect calorimetry in order to determinethe exact energy expenditure over a 24 hr period and then administer100% of the nutrients required to meet the energy expenditure of theindividual patient. However, a close evaluation of their data revealsthe percent contribution of glucose from gluconeogenesis indicates theirpatients were overfed. Despite the attempt to measure energy expenditureand deliver 100% of needed nutrients enterally, they inadvertentlyoverfed their patients. Overfeeding like underfeeding, of hospitalizedpatients can result in serious negative consequences includinginfection, prolonged ventilation, metabolic disturbances (hyperglycemia,dyslipidemia, liver dysfunction), morbidity and mortality (50).

Some Other Applications

Already described are patient treatments following trauma and chronicillness.

The invention also includes methods for managing those extreme and othercases and instances when assessment of BES, diagnosis and treatment areappropriate. Such examples include, but are not limited to assessing BESof patients or others before and after surgery, before and after drugtreatment or dietary intervention, or any situation in which knowing, orstandardizing BES is essential for determining outcome of any treatment,or establishing the effect of said treatment on humans or other mammals.

An example of using the new invention to set the BES background in whichto evaluate safety, efficacy and functionality in the process of drugdevelopment. Drug development is a term used to describe the process ofbringing a drug to market that includes pre-clinical research includinganimal studies, and human clinical trials, potentially leading toregulatory approval. In well-fed individuals % GNG can be as low as 10%,whereas after several days in the ICU % GNG could be 70% in TBIpatients. Given this 7-fold range in GNG flux, pharmaceuticalmanufacturers with drugs that can affect metabolic flux ratesseveral-fold would have a difficult time demonstrating effectiveness oftheir new drug. Therefore, with the present invention of being able todetermine, and nourish individuals to the point of controlling GNG andestablishing a stable background in which to evaluate effectiveness of anew drug, the patient treatments could be optimized. Also, costsassociated with testing for the effectiveness of new drugs could beminimized.

Additionally, the application of % GNG may also improve dose responseand efficacy for already established drugs or for new uses for drugsalready on the market. For example, the commonly used drug Decadron(Dexamethasone) may have a new application in post-surgery applicationsfor inflammation. However, it is anticipated that Decadron will affectthe metabolic function of the patient. This augmented metabolic functionmight cause the patient to become catabolic simply from theadministration of the drug and thus obviate the desiredanti-inflammation characteristics of the drug by causing an undesirableside effect. If this action were to happen, the application for the drugmight be interpreted to cause the patient to have a poor outcome.However, the outcome might be enhanced by proper nourishment using % GNGas a diagnostic. Under an appropriately nourished state, theadministration of Decadron could minimize inflammation, leading todesired affect of administration of the drug and potentially a betteroverall outcome for the patient.

Determination of % GNG and The Timing, Type and Amount of NutrientDelivery

Fractional gluconeogenesis (% GNG) will typically be determined onpatients in the morning so that the Part 1 measurement can be made,interpreted in Part 2, and the target GNG or % GNG achieved during thework day. By this schedule, it can be anticipated that GNG be determined3 times during the day, ideally morning, noon and evening. The morningmeasurement will provide important information on the patient'snutritional state through the evening and effectiveness on the ongoing,individual patient's needs for enteral nutrition and MCC infusion toachieve % GNG in the target range. Importantly, also, the morningmeasurement will inform clinicians to the need to adjust rates ofenteral nutrition and MCC infusion. The noon, or early afternoonmeasurement will be important to monitor the individual patient'schanging state, and to evaluate effectiveness of morning adjustments toachieve the target % GNG. And, the evening measurement will be importantto establish stability of blood glucose concentration and % GNG in thepatient during night hours when medical attention is typically lessfrequent.

Recovery from Illness and Trauma and Resumption of Oral Feeding

With the above described treatments (A-D) exquisite glycemic controlshall be accomplished in comatose patients. However, procedures need tobe in place for when patients recover consciousness sufficiently toallow the taking of some oral, including real-food, nutrition, butcontinue to require intravascular nutrient support. In all conditions,whether determined on patients after arising in the morning, or after a12-hr fast, the target % GNG of 25% remains in place. For example,because most dietary energy might be consumed during awake hours,parenteral nutrition may be insufficient to prevent GNG from exceeding25% of glucose Ra. Accordingly, food and parenteral nutrition would begiven to achieve the target range of −20-25% GNG.

Care of Infants

The exhaustive body of literature cited above deals largely with thecare of injured and ill adults. However, the CDC reports 500,000pre-term births annually in the US. Pre-term is the birth of an infantprior to 37 weeks of gestation. Pre-term birth is the most frequentcause of infant death, and leading cause of neurological disabilities(49). Such children are in obvious need of nutritive support, whether ornot surgery is required for survival, and the provision of nutritivesupport may avoid the development of conditions such as cerebral palsy,developmental delay, hearing impairment and other neurologicaldisabilities and death. Although not highly developed in the literature,the provision of nutritive support to premature infants, and themonitoring of gluconeogenesis have been subjects of investigation (43,77). Gluconeogenesis has been reported to be established 4-6 hr afterbirth in full-term children (43), and gluconeogenesis does respond tothe availability nutritive support (77). Extremely low birth weightpre-term infants do present abnormalities in the ability to regulate GNGin response to nutrient supply (19), but nutritive support isnone-the-less essential to the survival in such infants.

Care of Ill or Injured Mammals

Significant portions of the invention, principles, theory and practicewould likely apply to the care of most mammals (11, 23) and others (37,72, 84). A notable exception will apply to the care of ruminant mammalswhose gut is evolved to digest fibrous plant materials such as celluloseto propionate (42), which is closely related to lactate(2-hydroxypropionate). Hence, in the case of ruminant mammals, thenutritional formulation could include 0-100% propionate.

Nutritive Formulations

Nutritive formulations to be given to patients after estimation of GNG(and in some cases, other biomarkers as well), has been discussed above,but will be discussed in more detail below.

Lactate-Based Formulations (Sanguisal-L)

In its simplest form, the MCC cocktail would be sodium-L-(+)-Lactateprepared by titrating L-(+)-Lactic acid with NaOH. The followingdescribe such procedures: (24, 52, 55-57). Briefly, the MCC infusioncocktail is prepared by mixing concentrated (30-88%) L-(+)-lactic acidsolution (e.g., Sigma-Aldrich or PCCA) in 2 N NaOH to pH 4.8. In theexample provided, the starting point is a 30% stock lactic acidsolution: 300 g 30% lactic acid stock solution is titrated with 133.3 g2N NaOH and diluted to 1,000 ml with water. This will produce a 11.2%weight-by-volume (“w/v”) MCC cocktail (sodium-L-(+)-Lactate) with anosmolality approximating 2,000 mOsm/l. Depending on the stock lacticacid solution used, a NaOH titrating solution >2 N may be necessary toneutralize stock lactic acid without exceeding the intended volume.Regardless of the specifics of acid titration, a 1.72% Nat-lactateaqueous solution is isosmotic (308 mOsm/l); consequently, as initiallymixed the above described MCC is far too concentrated to be given in aperipheral vein and will need to be diluted to less than 1,000 mOsm/l (a5.6% Nat-lactate solution). In a peripheral arm vein and a 5.0-5.6%Nat-lactate MCC should be given with normal (0.9%, 308 mOsm/l) saline oranother isosmotic solution such as 5 mM glucose (dextrose) in water,commonly called D5W (see more below).

This isosmotic admixture of MCC and diluent at the MCC infusion sitewill be sufficient to maintain vessel patency and prevent phlebitis orhemolysis at the infusion site (56). In a large vessel a 5% Na⁺-lactateMCC cocktail could be given without expectation of hemolysis at theinfusion site (57). Regardless of the dilution, the initial MCC solutiondelivery rate should be adjusted to deliver 10-50 (micro Moles)μMoles/kg/min, with maintenance infusion rate targeting blood lactateconcentration of 3.5-4.5 mM, although higher levels (6 mM) have beenused without ill effects (57, 71). Consistent with section methodsdescribed above, an infusion rate of 11 μMoles/kg/min would deliver themass equivalent of 1.0 mg/kg/min of glucose, and infusion rate of 23μMoles/kg/min of sodium lactate MCC would deliver the mass equivalent of3 mg/kg/min glucose, and an infusion rate of 50 μMoles/kg/min woulddeliver the mass equivalent of 4.5 mg/kg/min of glucose. Ideally, theMCC is prepared from highest purity materials, is pathogen free,certified for human pharmaceutical use and is delivered into a largecentral vein, but peripheral vein can be used if administered withphysiological saline to minimize osmolality and pH effects at theinfusion site that might provoke phlebitis of hemolysis.

In another version of the simplest form, the MCC cocktail would besodium-L-(+)-Lactate prepared from the dry, powdered salt in deionizedwater to the concentration intended [e.g., for an isosmotic solution:154 mM lactate (plus 154 mM Na⁺) total osmolarity 308 mOsm/l], andinfused in the above-stated amounts to raise arterial [lactate] to theintended levels.

In the preferred form of the simplest iteration, the basicsodium-L-(+)-Lactate cocktail would be amended to include other lactatesalts as exist in the plasma of healthy humans. Sodium ion (Na⁺) is themain cation in plasma, normally 145 mM, and values for K+, Ca⁺⁺, andMg⁺⁺ are, respectively, 4, 2.5 and 1.5 mM. Hence, a mixture of inorganiclactate salts comprised of Na⁺-, K⁺-, Ca⁺⁺-, and Mg⁺⁺-lactate would becombined in the ratio of 144, 4, 2.5, and 1.5. In this embodiment ofinvention, the main anion would be lactate, but phosphates are importantions in energy metabolism and would be added in the form of 1 mMNaH₂PO₄—. Because Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, and H₂PO₄— are present in theplasma of healthy individuals at levels of 145, 4, 2.5, 1.5, and 1.0milliequivalent per liter (mEq/l) (53). This particular embodiment ofMCC could be termed “Sanguisal” from the Latin words for blood (sanguis)and salt. To reiterate from above, the provision of sodium and othercations as a means to deliver lactate anions in an MCC has the advantageof reducing brain swelling following TBI (15) as well as providingnutritive support to intensive care patients following trauma (70).

Pyruvate-Based Formulations (Sanguisal-P)

As an alternative to Sanguisal-L that uses lactate as the major anion(vide supra), Sanguisal-P will involve the use of pyruvate (P) as themajor anion, while at the same time maintaining the above-stated levelsof cations {Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, and H₂PO₄— as are present in the plasmaof healthy individuals: 145, 4, 2.5, 1.5, and 1.0 mEq/l}. Like itscounterpart lactate, pyruvate is the precursor to lactate in glycolysis.Further, pyruvate is an oxidizable fuel and GNG precursor whosemetabolism may affect cell redox status if converted to lactate.Further, pyruvate possesses antioxidant properties (48). Althoughtypically present in 1/10 or lesser concentration compared to lactate,pyruvate has been introduced into the systemic circulation of largemammals in which the circulation has been interrupted to mimic cardiacarrest (61-63, 68). In such cases 100% pyruvate infusion raises thecirculating pyruvate level, but more so, the circulating lactate levelachieving a circulating L/P of 2-3. None the less, because of itschemical structure, pyruvate has the advantage over lactate of servingas an antioxidant in the myocardium subjected to reperfusion injury,and, by extension exogenous pyruvate my also serve to scavenge freeradicals in the brain after blood flow is interrupted. A problem withregard to the use of Sanguisal-P, in contrast to the Sanguisal-L form,is that in circulation pyruvate is rapidly converted to lactate due tothe effects of lactate dehydrogenase in red blood cells and the lungparenchyma (41). Hence, for the antioxidant properties of pyruvate, theoptimal site of Sanguisal-P infusion would be in the carotid artery orascending aorta. Still, by the ability to infuse mixtures of Sanguisal-Land Sanguisal-P into the systemic circulation, ascending or carotidartery, the clinician would have the opportunity to affect redox statusin an injured brain. However, because carotid or ascendingcatheterization is uncommon even in the ICU, systemic solutions of avery low L/P ratio (i.e., high P/L) such as infusion of 100%Sanguisal-P, or a mixture of Sanguisal-P/Sanguisal-L of 2.0 (L/P=½ inthe infusate), would serve to raise the arterial level of pyruvaterelative to that of lactate.

In its simplest form, Sanguisal-P would be sodium-L-(+)-Pyruvateprepared by titrating L-(+)-Pyruvic acid with NaOH as described abovefor lactate (24, 52, 55-57). As with lactate (vide supra), the initialNa⁺-pyruvate infusion rate would deliver 11-50 (micro Moles)μMoles/kg/min, with maintenance infusion rate targeting blood lactateconcentration of 3.5-4.5 mM, although higher levels (6 mM) have beenused without ill effects (57, 71). Consistent with section methodsdescribed above, infusion rate of 11 μMoles/kg/min of sodium pyruvateMCC would deliver the mass equivalent of 1.0 mg/kg/min of glucose, aninfusion rate of 23 μMoles/kg/min would deliver the mass equivalent of 3mg/kg/min glucose, whereas an infusion rate of 50 μMoles/kg/min ofsodium pyruvate MCC would deliver the mass equivalent of 4.5 mg/kg/minof glucose. Ideally, the Na⁺-pyruvate MCC is prepared from highestpurity materials, is pathogen free, certified for human pharmaceuticaluse and is delivered into a large central vein, but peripheral vein canbe used if administered with physiological saline to minimize osmolalityand pH effects at the infusion site that might provoke phlebitis ofhemolysis.

In another version of the simplest form, the MCC cocktail would besodium-L-(+)-Pyruvate prepared from the dry, powdered salt in deionizedwater to the concentration intended, and infused in the above-statedamounts to raise arterial [lactate] to the intended levels. Toreiterate, manufacturing and storing pyruvate-containing formulationssuch as Sanguisal P and Sanguisal L-P mixtures should ve anhydrous. Puresterile water can be added immediately before delivery to avoid pyruvatedegradation and accumulation of undesired pyruvate degradation products.

In the preferred form of the simplest iteration, the basicsodium-L-(+)-Pyruvate cocktail would be amended to include otherpyruvate salts as exist in the plasma of healthy humans. Sodium ion(Na⁺) is the main cation in plasma, normally 145 mM, and values for K⁺,Ca⁺⁺, and Mg⁺⁺ are, respectively, 4, 2.5 and 1.5 mM. Hence, a mixture ofinorganic lactate salts comprised of Na⁺-, K⁺-, Ca⁺⁺-, and Mg⁺⁺-lactatewould be combined in the ratio of 144, 4, 2.5, and 1.5. In thisembodiment of invention, the main anion would be pyruvate, butphosphates are important ions in energy metabolism and could be added inthe form of 1 mM NaH₂PO₄—. Because Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, and H₂PO₄— arepresent in the plasma of healthy individuals at levels of 145, 4, 2.5,1.5, and 1.0 milliequivalent per liter (mEq/l, this particularembodiment of MCC could be termed “Sanguisal” from the Latin words forblood (sanguis) and salt (53); in this case Sanguisal-P (for pyruvate).To reiterate from above, the provision of pyruvate and other cations asa means to deliver lactate anions in an MCC has the advantage ofreducing brain swelling following TBI, as has been observed by theinventors and other researchers in studies to be published in the comingmonths, see also (15). The invention can also provides nutritive supportto intensive care unit patients following trauma (70), and acting as areactive oxygen species (“ROS”) scavenger (48).

In this preferred form, Sanguisal-P would be prepared from the dry,powdered pyruvate salts (Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, and H₂PO₄—) in the ratiosof 145:4:2.5:1.5:1.0 in sterile deionized water to an anionconcentration of 154 milliequivalent per liter (mEq/l), and infused inthe above-stated amounts to raise arterial [pyruvate] to 1-2 mM and anarterial [lactate] in the intended range.

Lactate and Pyruvate Combination-Based Formulations (Sanguisal-L/P)

Isosmotic (154 mM) mixtures of Sanguisal-L and -P may be mixed and usedto change the circulating L/P. Infusions of 2N Na⁺-Pyruvate into swinewere observed to raise arterial [pyruvate] to 3.5 mM and [lactate] to 8mM for an L/P in the range of 2-3 (68). If an arterial L/P of 10-11 istypical of healthy resting humans (36), then infusing a Sanguisal mix of2 parts Sanguisal-P to 1 Part Sanguisal-L may reduce an elevatedsystemic L/P to that seen in healthy individuals. Then, by observation,the clinician has the ability to lower the circulating L/P by loweringthe L/P of the Sanguisal mix given. Conversely, the clinician has theability to raise the circulating L/P by increasing the L/P of theSanguisal mix given. In summary, Sanguisal (S) mixes of 100% S-Pyruvateto 100% S-Lactate may be used to provide nutritive support to theinjured brain and other injured or non-injured tissues, provide agluconeogenic precursor, and scavenge ROS in all tissues perfused.

With regard to the infusion of Sanguisal-P, one may logically ask: “whyis the resulting arterial [lactate] monitored?” There are practical aswell as scientific reasons for this. From the practical standpoint,rapid lactate, but not pyruvate analyzers are available. Andscientifically, lactate is the preferred monocarboxylate compound (MCC)in nature: the L/P in arterial blood of healthy individuals is minimally10, and rises more than an order of magnitude in normal physiology.Secondly, pyruvate is rapidly converted to lactate in the blood by theaction of lactic dehydrogenase in erythrocytes (RBCs) in the blood (69)and the lung parenchyma (41), and lactate, not pyruvate, it the majorfuel source and GNG precursor (see above).

Regardless of whether Sanguisal-L, -P or -L/-P mixtures areadministered, the clinician will monitor % GNG and titrate the Sanguisalinfusion rate with the target to achieving approximate estimates of GNGbetween 15-30%.

Lactate Esters as MCCs

As described herein, Sanguisal-L and -P are inorganic salt-based meansto deliver nutritive support. However, it is possible to deliver lactateand other nutritive compounds (pyruvate and acetate) by other means,including esters. Arginyl lactate (U.S. Pat. No. 5,420,107) has beenextensively used as an (enteral) amendment to sports drinks to provideenergy and blood buffering (1, 25). Arginyl lactate is formed by theelectrostatic binding of lactate from lactic acid and the basic(zwitterion) amino acid arginine under basic conditions. Theseindividual units dissociate spontaneously at neutral pH as exists inhuman plasma. The components of arginyl lactate (arginine and lactate)are benign and efficacious in human blood.

Similarly, the lactate thiolester formed from the combination of lactateand N-acetylcysteine (called “LNACE”, see, for example, U.S. Pat. No.6,482,853) has been proposed as an amendment to sports drinks to provideenergy and blood buffering. Like arginyl lactate, LNACE is yet anotherplatform for parenteral nutrition of an ill or injured patient.

Still another means to deliver parenteral nutrition of an ill or injuredpatient is glycerol tri-lactate (called “GTL”, see, for example, U.S.Pat. No. 6,743,821). Glycerol tri-lactate is formed by theesterification of glycerol by lactic acid by means of organic orenzymatically catalyzed processes (see, for example, U.S. Pat. No.6,743,821). These individual units rapidly dissociate because of thelipases and esterases in human plasma. The components of GTL (glyceroland lactate) are benign and efficacious in human blood. Glycerol hasbeen used as a plasma expander (76) and gluconeogenic precursor (73). Interms of nutrient delivery, GTL is preferred over sodium- and otherinorganic salts of lactate because more lactate is carried, no sodiumload is incurred, and because the glycerol carrier is efficacious.

Still another means to deliver parenteral nutrition of an ill or injuredpatient is glycerol tri-acetate (called “GTA”, see, for example, U.S.Pat. No. 6,743,821), or acetin. Acetate is another body, although notbrain, fuel energy source.

Lactate, Pyruvate and Dextrose Combination-Based Formulations(Sanguisal-L/P/D)

State of the art is to provide parenteral nutritive support to patientsusing 5% dextrose (D5W, glucose in water, vide supra). Because glucose(dextrose) has a molecular weight of 180, osmolality of D5W is 278 mM,which in the low range of normal plasma osmolality (275-310 mEq/l). Eventhough the glucose concentration in D5W is 50 times greater thanhomeostatic in plasma, in terms of its isosmotic effect with glucosealone being the only solute, D5W is isosmotic. Mixing equal isosmoticsolutions such as equal volumes of 154 mM Sanguisal-L and D5W glucosewill produce an osmolality in the high end of the normal range. As notedearlier, this slightly elevated osmolality because of sodium contentwill draw fluid from tissues into the vascular compartment, thusmitigating swelling due to injury (15).

Lactate, Pyruvate, and Amino Acid Combination-Based Formulations(Sanguisal-L/P/A)

Parenteral nutrition is provided to hasten nutrient delivery orgastro-intestinal and other conditions exist to limit the enteraldelivery of nutrients. The above-identified Sanguisal formulations areall carbohydrate (CHO)-based and can be used and adjusted to nourish theinjured brain and other organs. In vivo, energy balance and nitrogenbalance interact, the RDA for amino acid and protein intake being 0.8g/kg body weight/day (8, 51), with the assumption being that the dietsupplies sufficient energy to maintain energy balance. Although thelatter assumption is seldom recognized with athletes taking upwards of 2g/lb/body weight (60), even very high protein diets that are accompaniedby a very high nitrogen load, will lack energy to maintain a person innitrogen balance. Accordingly, in attempts to maintain nitrogen balanceand body mass in injured and ill patients, amino acid and proteinsupplementation to the extent of 50% above the RDA for amino acids andproteins on the background of adequate energy supply is deemed prudent(82).

Commercially available parenteral amino acid solutions contain essentialand non-essential amino acids. In contemporary literature, investigatorshave evaluated effects of emphasizing particular amino acids, and typesof amino acids including: glutamine, arginine, cysteine, taurine, andbranched chain amino acids (“BCAA”, leucine, isoleucine, and valine,particularly leucine). At present it is clear that parenteral solutionscontaining mixes of essential and non-essential amino acids are safe andefficacious, being part of routine parenteral nutrition. Such solutionsare typically hyperosmotic, but less than 1,000 mOsm/l. As such, currentart allows that infusions of Sanguisal-L, Sanguisal-P, or mixes ofSanguisal (L/P), could be augmented by infusions of amino acidparenteral solutions delivering 1.0-1.2 g nitrogen/kg body weight/day.

Lactate, Pyruvate, Dextrose and Amino Acid Combination-BasedFormulations (Sanguisal-L/P/D/A)

When % GNG is unknown, with the assumption that blood glucose can bemonitored in real time, a clinician can provide parenteral and enteralnutrition as described above. In the event of hepatic or renal failure,and consequent limitations in a patient's ability to clear sodium orregulate glycemia by means of providing GNG precursors, a clinician maymoderate the course of providing lactate- and, or, pyruvate-based MCCs,and instead supplement the patient with D5W, in extreme hypoglycemia,D10W (5 mM glucose (dextrose)).

Adding D₂O to Sanguisal Formulations

For assessment of GNG using the D₂O method, first responders will needto take a first (“background”) blood sample before the injection of D₂O.As noted above, the 0.3 to 0.5% D₂O abundance necessary to estimate GNGfrom the penta-acetate derivative of glucose and GC/MS could be obtainedby a bolus of D₂O. However, also as noted above, the 0.3 to 0.5%abundance of D₂O could be maintained over days and weeks by adding D₂Oto all infusates given to TBI and other injured and ill patients. AsSanguisal solutions will be given to provide parenteral nutrition andmaintain glycemia, as a preferred form, D₂O could be to make Sanguisalsolutions 0.3 to 0.5% D₂O. Another preferred form of D₂O could be tomake common intravenous saline solutions 0.3 to 0.5% D₂O as the majorityof the exogenous fluids delivered to an ill or injured patient comesform the intravenous (“i.v.”) saline solutions routinely used in thehospital. If the exogenous fluid load can be controlled by the clinicianthrough use of the common i.v. saline solution, then the enrichment ofbody water by deuterium can also be controlled and therefore themeasurement of % GNG can be made for the duration of the stay at thehospital as frequently as required by the attending clinician. Forexample, if 100% of the exogenous solution come from the i.v. salinesolution and the i.v. solution is a 1 liter bag, the i.v. solution willhave approximately 0.3 to 0.5 g of D₂O added. If enteral feeding, forexample, contributes 25% of the exogenous fluids, then the D₂O salinesolution could contain 25% more D₂O to accommodate for the increasedingestion of exogenous fluids. Therefore the saline solution would nowcontain approximately 0.375 to 0.625 g D₂O.

With regard to the present invention, the many features and advantagesof the present invention are apparent from the written description, andthus, it is intended by the appended claims to cover all such featuresand advantages of the invention. Further, since numerous modificationsand changes will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and operationas illustrated and described. Hence, all suitable modifications andequivalents may be resorted to as falling within the scope of theinvention.

Various elements of the invention are described as modules implementedas software on a general purpose computer and others as hardwareelements. It should be apparent that in various embodiments of theinvention, implementation of software can be executed by embeddedhardware, or vice versa, or in some combination of software andhardware. Also, a computer may take the form of an integrated circuit,printed circuit board, handheld computer, or any general-purposecomputer without limitation.

Part of the invention may be implemented by a general-purpose computer,embedded circuitry, or some combination of these. The software executionmay be accomplished through the use of a program storage device readableby the computer and encoding a program of instructions executable by thecomputer for performing the operations described above. The programstorage device may take the form of any memory known in the art orsubsequently developed. The program of instructions may be object code,i.e., in binary form that is executable more-or-less directly by thecomputer; in source code that requires compilation or interpretationbefore execution; or in some intermediate form such as partiallycompiled code and/or a collection of executable library files. Theprecise forms of the program storage device and of the encoding ofinstructions are immaterial here.

The invention also contemplates use of computer networks known in theart, including but not limited to, intranets such as corporate networks,local and wide area networks, the Internet and the World Wide Web. Wireand wireless communication and communication protocols known in the art,such as, but not limited to, radio, infrared, Bluetooth, Ethernet andother wireless and wired networks, are also contemplated.

Preferred embodiments of flow direction between elements, looping anditeration are discussed, but alternative embodiments of these flows arecontemplated by the invention. Any elements or other features describedin the figures, even if not described in the specification, aresupported in the figures so as to be enabling. All references cited hereare incorporated in their entirety for all purposes.

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What is claimed is: 1.-30. (canceled)
 31. A method for estimating thefractional gluconeogenesis of a patient, the method comprising: (a)administering a label to the patient; (b) taking a blood sample from thepatient; (c) analyzing glucose or a glucose derivative from the bloodsample; (d) obtaining a value for fractional gluconeogenesis; (e)obtaining a value for fractional gluconeogenesis plus glycogenolysis;and (f) using (d) and (e) to estimate fractional gluconeogenesis. 32.The method of claim 31 wherein the label is deuterium.
 33. The method ofclaim 31 wherein less than about 1% of the body water is labeled. 34.The method of claim 31 wherein the body water is labeled with an initialbolus or ongoing infusion or both.
 35. The method of claim 31 whereinthe value in (e) also represents % body water labeled.
 36. The method ofclaim 31 wherein the glucose derivative is a penta-acetate glucosemolecule with molecular weight of about
 390. 37. The method of claim 31wherein (d) is based on the abundance of the label on one or more ofglucose carbons 1, 3, 4, 5,
 6. 38. The method of claim 31 wherein (e) isbased on the abundance of the label on glucose carbon
 2. 39. The methodof claim 31 wherein glucose Ra is estimated to further provide anestimate of absolute rate of gluconeogenesis.
 40. The method of claim 31wherein the analyzing is done using a mass spectrometer.
 41. The methodof claim 31, wherein the analyzing is done using one or morechromatography techniques.
 42. The method of claim 31, wherein uponestimating the fractional gluconeogenesis, the patient is administered aparenteral or enteral nutritive formulation or both.
 43. The method ofclaim 31 wherein the formulation contains GNG precursor or MCC or both.44. The method of claim 31 wherein the formulation contains pyruvate orlactate or both.
 45. The method of claim 31 wherein the formulation isadministered or increased if the estimated fractional gluconeogenesis isabove about 25%.
 46. The method of claim 31 wherein the formulation isadministered or increased if the estimated fractional gluconeogenesis isabove about 35%.
 47. The method of claim 31 wherein the formulation isstopped or decreased if the estimated fractional gluconeogenesis isbelow about 15%.
 48. The method of claim 31 wherein the formulation isstopped or decreased if the estimated fractional gluconeogenesis isbelow about 20%.
 49. A method for providing nutritional support to apatient, the method comprising: (a) administering a label to thepatient; (b) taking a blood sample from the patient; (c) analyzingglucose or a glucose derivative from the blood sample; (d) obtaining avalue for fractional gluconeogenesis; (e) obtaining a value forfractional gluconeogenesis plus glycogenolysis; (f) using (d) and (e) tocreate to estimate fractional gluconeogenesis; and (g) administering aparenteral nutritive formulation or enteral nutritive formulation orboth to the patient based upon the fractional gluconeogenesis estimate.50. The method of claim 49 wherein the label is deuterium.
 51. Themethod of claim 49 wherein less than about 1% of the body water islabeled.
 52. The method of claim 49 wherein the body water is labeledwith an initial bolus or ongoing infusion or both.
 53. The method ofclaim 49 wherein the value in (e) also represents % body water labeled.54. The method of claim 49 wherein the glucose derivative is apenta-acetate glucose molecule with molecular weight of about
 390. 55.The method of claim 49 wherein the value in (d) is based on theabundance of the label on one or more of glucose carbons 1, 3, 4, 5, 6.56. The method of claim 49 wherein the value in (e) is based on theabundance of the label on glucose carbon
 2. 57. The method of claim 49wherein the formulation contains GNG precursor or MCC or both.
 58. Themethod of claim 49 wherein the molecule is analyzed using a massspectrometer.
 59. A method for estimating the fractional gluconeogenesisof a patient, the method comprising: (a) administering an amount oflabel to the patient; (b) estimating the fraction of body water that hasbeen labeled; (c) using the estimating in (b) to create a baseline forthe amount of total glucose production; (d) estimating an amount ofglucose production only from gluconeogenesis by measuring the label; and(e) using (c) and (d) to estimate the patient's fractionalgluconeogenesis.
 60. A method for providing nutritional support to apatient, the method comprising: (a) administering an amount of label tothe patient; (b) estimating the fraction of body water that has beenlabeled; (c) using the estimating in (b) to create a baseline for theamount of total glucose production; (d) estimating an amount of glucoseproduction only from gluconeogenesis by measuring the label; (e) using(c) and (d) to estimate the patient's fractional gluconeogenesis; and(f) administering a parenteral nutritive formulation to the patientbased upon the fractional gluconeogenesis estimate.